Fluoroalkoxy-substituted 1,3-dihydro-isoindolyl compounds and their pharmaceutical uses

ABSTRACT

The invention encompasses novel compounds, pharmaceutically acceptable salts, hydrates, solvates, clathrates, enantiomers, diastereomers, racemates, or mixtures of stereoisomers thereof, pharmaceutical compositions of these compounds, and methods of using these compounds and compositions in mammals for treatment or prevention of diseases associated with PDE4.

This application is a divisional of the U. S. application Ser. No.12/363,664, filed Jan. 30, 2009, now U.S. Pat. No. 7,893,102 allowed,which is a divisional of the U.S. application Ser. No. 11/601,355, filedNov. 16, 2006, now U.S. Pat. No. 7,504,427, issued Mar. 17, 2009, whichis a divisional of the U.S. application Ser. No. 10/748,085, filed Dec.29, 2003, now U.S. Pat. No. 7,173,058, issued Feb. 6, 2007, which claimsthe benefit of U.S. Provisional Application No. 60/436,975, filed Dec.30, 2002, each of which is incorporated herein by reference in itsentirety.

1. FIELD OF THE INVENTION

The invention encompasses novel fluoroalkoxy-substituted1,3-dihydro-isoindolyl compounds, pharmaceutical compositions of thesecompounds, and methods of using these compounds and compositions inmammals for treatment or prevention of diseases associated with PDE4inhibition, abnormal TNF-α levels and/or MMP inhibition.

2. BACKGROUND OF THE INVENTION

2.1 TNF-α

Tumor necrosis factor alpha (TNF-α) is a cytokine that is releasedprimarily by inflammation and mononuclear phagocytes in response toimmunostimulators. TNF-α is capable of enhancing most cellularprocesses, such as differentiation, recruitment, proliferation, andproteolytic degradation. At low levels, TNF-α confers protection againstinfective agents, tumors, and tissue damage. However, TNF-α also hasrole in many diseases. When administered to mammals such as humans,TNF-α causes or aggravates inflammation, fever, cardiovascular effects,hemorrhage, coagulation, and acute phase responses similar to those seenduring acute infections and shock states. Enhanced or unregulated TNF-αproduction has been implicated in a number of diseases and medicalconditions, for example, cancers, such as solid tumors and blood-borntumors; heart disease, such as congestive heart failure; and viral,genetic, inflammatory, allergic, and autoimmune diseases.

Cancer is a particularly devastating disease, and increases in bloodTNF-α levels are implicated in the risk of and the spreading of cancer.Normally, in healthy subjects, cancer cells fail to survive in thecirculatory system, one of the reasons being that the lining of bloodvessels acts as a barrier to tumor-cell extravasation. However,increased levels of cytokines have been shown to substantially increasethe adhesion of cancer cells to endothelium in vitro. One explanation isthat cytokines, such as TNF-α, stimulate the biosynthesis and expressionof a cell surface receptors called ELAM-1 (endothelial leukocyteadhesion molecule). ELAM-1 is a member of a family of calcium-dependentcell adhesion receptors, known as LEC-CAMs, which includes LECAM-1 andGMP-140. During an inflammatory response, ELAM-1 on endothelial cellsfunctions as a “homing receptor” for leukocytes. ELAM-1 on endothelialcells was shown to mediate the increased adhesion of colon cancer cellsto endothelium treated with cytokines (Rice et al., 1989, Science246:1303-1306).

Inflammatory diseases such as arthritis, related arthritic conditions(e.g., osteoarthritis and rheumatoid arthritis), inflammatory boweldisease, sepsis, psoriasis, chronic obstructive pulmonary diseases andchronic inflammatory pulmonary diseases are also prevalent andproblematic ailments. TNF-α plays a central role in the inflammatoryresponse and the administration of their antagonists block chronic andacute responses in animal models of inflammatory disease.

Enhanced or unregulated TNF-α production has been implicated in viral,genetic, inflammatory, allergic, and autoimmune diseases. Examples ofsuch diseases include, but are not limited to: HIV; hepatitis; adultrespiratory distress syndrome; bone-resorption diseases; chronicobstructive pulmonary diseases; chronic pulmonary inflammatory diseases;dermatitis; cystic fibrosis; septic shock; sepsis; endotoxic shock;hemodynamic shock; sepsis syndrome; post ischemic reperfusion injury;meningitis; psoriasis; fibrotic disease; cachexia; graft versus hostdisease (GVHD); graft rejection; auto-immune disease; rheumatoidspondylitis; arthritic conditions, such as rheumatoid arthritis,rheumatoid spondylitis and osteoarthritis; osteoporosis;inflammatory-bowel disease; Crohn's disease; ulcerative colitis;multiple sclerosis; systemic lupus erythrematosus; ENL in leprosy;radiation damage; asthma; and hyperoxic alveolar injury. Tracey et al.,1987, Nature 330:662-664 and Hinshaw et al., 1990, Circ. Shock30:279-292 (endotoxic shock); Dezube et al., 1990, Lancet, 335:662(cachexia); Millar et al., 1989, Lancet 2:712-714 and Ferrai-Balivieraet al., 1989, Arch. Surg. 124:1400-1405 (adult respiratory distresssyndrome); Bertolini et al., 1986, Nature 319:516-518, Johnson et al.,1989, Endocrinology 124:1424-1427, Holler et al., 1990, Blood75:1011-1016, and Grau et al., 1989, N. Engl. J. Med. 320:1586-1591(bone resorption diseases); Pignet et al., 1990, Nature, 344:245-247,Bissonnette et al., 1989, Inflammation 13:329-339 and Baughman et al.,1990, J. Lab. Clin. Med. 115:36-42 (chronic pulmonary inflammatorydiseases); Elliot et al., 1995, Int. J. Pharmac. 17:141-145 (rheumatoidarthritis); von Dullemen et al., 1995, Gastroenterology, 109:129-135(Crohn's disease); Duh et al., 1989, Proc. Nat. Acad. Sci. 86:5974-5978,Poll et al., 1990, Proc. Nat. Acad. Sci. 87:782-785, Monto et al., 1990,Blood 79:2670, Clouse et al., 1989, J. Immunol. 142, 431-438, Poll etal., 1992, AIDS Res. Hum. Retrovirus, 191-197, Poli et al. 1990, Proc.Natl. Acad. Sci. 87:782-784, Folks et al., 1989, PNAS 86:2365-2368 (HIVand opportunistic infections resulting from HIV).

2.2 PDE4

Adenosine 3′,5′-cyclic monophosphate (cAMP) also plays a role in manydiseases and conditions, such as, but not limited to asthma andinflammation (Lowe and Cheng, Drugs of the Future, 17(9), 799-807,1992). It has been shown that the elevation of cAMP in inflammatoryleukocytes inhibits their activation and the subsequent release ofinflammatory mediators, including TNF-α and nuclear factor κB (NF-κB).Increased levels of cAMP also lead to the relaxation of airway smoothmuscle.

It is believed that primary cellular mechanism for the inactivation ofcAMP is the breakdown of cAMP by a family of isoenzymes referred to ascyclic nucleotide phosphodiesterases (PDE) (Beavo and Reitsnyder, Trendsin Pharm., 11, 150-155, 1990). There are twelve known members of thefamily of PDEs. It is recognized that the inhibition of PDE type IV(PDE4) is particularly effective in both the inhibition of inflammatorymediated release and the relaxation of airway smooth muscle (Verghese,et al., Journal of Pharmacology and Experimental Therapeutics, 272(3),1313-1320, 1995). Thus, compounds that specifically inhibit PDE4 mayinhibit inflammation and aid the relaxation of airway smooth muscle witha minimum of unwanted side effects, such as cardiovascular oranti-platelet effects.

The PDE4 family that is specific for cAMP is currently the largest andis composed of at least 4 isozymes (a-d), and multiple splice variants(Houslay, M. D. et al. in Advances in Pharmacology 44, eds. J. August etal., p. 225, 1998). Totally there may be over 20 PDE4 isoforms expressedin a cell specific pattern regulated by a number of different promoters.Disease states for which selective PDE4 inhibitors have been soughtinclude: asthma, atopic dermatitis, depression, reperfusion injury,septic shock, toxic shock, endotoxic shock, adult respiratory distresssyndrome, autoimmune diabetes, diabetes insipidus, multi-infarctdementia, AIDS, cancer, Crohn's disease, multiple sclerosis, cerebralischemia, psoriasis, allograft rejection, restenosis, ulceratiavecolitis, cachexia, cerebral malaria, allergic rhino-conjunctivitis,osteoarthritis, rheumatoid arthrirtis, chronic obstructive pulmonarydisease (COPD), chronic bronchitis, cosinophilic granuloma, andautoimmune encephalomyelitis (Houslay et al., 1998). PDE4 is present inthe brain and major inflammatory cells and has been found in abnormallyelevated levels in a number of diseases including atopic dermatitis oreczema, asthma, and hay fever among others (reference OHSU flyer and J.of Allergy and Clinical Immunology, 70: 452-457,1982 by Grewe et. al.).In individuals suffering from atopic diseases elevated PDE-4 activity isfound in their peripheral blood mononuclear leukocytes, T cells, mastcells, neutrophils and basophils. This increased PDE activity decreasescAMP levels and results in a breakdown of cAMP control in these cells.This results in increased immune responses in the blood and tissues ofthose that are affected.

Clinical use of inhibitors of PDE 4 have shown them to be broad spectrumanti-inflammatory agents with impressive activity in models of asthma,chronic obstructive pulmonary disorder (COPD) and other allergicdisorders such as atopic dermatitis and hay fever. PDE 4 inhibitors thathave been used include theophylline, rolipram, denbufylline, ARIFLO,ROFLUMILAST, CDP 840 (a tri-aryl ethane) and CP80633 (a pyrimidone).PDE4 inhibitors have been shown to influence eosinophil responses,decrease basophil histamine release, decrease IgE, PGE2, IL 10synthesis, and decrease anti-CD3 stimulated II-4 production. Similarly,PDE4 inhibitors have been shown to block neutrophil functions.Neutrophils play a major role in asthma, chronic obstructive pulmonarydisorder (COPD) and other allergic disorders. PDE4 inhibitors have beenshown to inhibit the release of adhesion molecules, reactive oxygenspecies, interleukin (IL)-8 and neutrophil elastase, associated withneutrophils which disrupt the architecture of the lung and thereforeairway function. PDE inhibitors influence multiple functional pathways,act on multiple immune and inflammatory pathways, and influencesynthesis or release of numerous immune mediators. J. M. Hanifin and S.C. Chan, Atopic Dermatitis-Therapeutic Implication for NewPhosphodiesterase Inhibitors, Monocyte Dysregulation of T Cells in AACINews, 7/2, 1995; J. M. Hanifin et al., Type 4 PhosphodiesteraseInhibitors Have clinical and In Vitro Anti-inflammatory Effects inAtopic Dermatitis, Journal of Investigative Dermatology, 1996, 107, pp51-56).

The first generation of PDE-4 inhibitors have been effective ininhibiting PDE4 activity and alleviating a number of the inflammatoryproblems caused by over expression of this enzyme. However, theireffectiveness has been limited by side effects, particularly when usedsystemically, of nausea and vomiting (Huang et al. Curr. Opin. In Chem.Biol. 2001, 5:432-438). Indeed, all of the PDE-4 inhibitors developed todate have been small molecule compounds with central nervous system andgastrointestinal side effects, i.e., headache, nausea/emesis, andgastric secretion.

2.3 MMP

Matrix metalloproteinases (MMPs) are a family of proteases (enzymes)involved in the degradation and remodeling of connective tissues.Excessive degradation of extracellular matrix by MMPs is implicated inthe pathogenesis of many diseases, including rheumatoid arthritis,osteoarthritis, cancer, multiple sclerosis, bone resorptive diseases(such as osteoporosis), chronic obstructive pulmonary disease,restenosis, cerebral hemorrhaging associated with stroke, periodontaldisease, aberrant angiogenesis, tumor invasion and metastasis, cornealand gastric ulceration, ulceration of skin, aneurysmal disease, and incomplications of diabetes. MMP inhibition is, therefore, recognized as agood target for therapeutic intervention of this type of diseases. Manycompounds having MMP inhibition activities have been reported (R. A.Nigel et al, Current Opinion on Therapeutic Patents, Vol. 4, 7-16,(1994), R. P. Beckett et al, Drug Discovery Today, Vol. 1, 16-26,(1996)). However, most of them are peptide derivatives designed based onthe amino acid sequence of the enzymatic cleavage site in the collagenmolecule constituting the substrate of MMP, thus a need exists for asmall molecule inhibitor of MMP.

Accordingly, there remains a need in the art for PDE4 inhibitors,compounds which regulate TNF-α production and which inhibit MMPproduction. In particular, there remains a need for inhibitors whichhave in vivo activity without or with reduced side effects.

3. SUMMARY OF THE INVENTION

The present invention provides compounds which are useful in thetreatment of diseases mediated by the inhibition of PDE4 as well asdiseases mediated by TNF-α and MMP. The invention also providespharmaceutical compositions comprising these compounds and methods ofusing the subject compounds and compositions for the treatment of avariety of diseases.

The compounds provided herein have the formula (I):

wherein:

Y is —C(O)—, —CH₂, —CH₂C(O)—, —C(O)CH₂—, or SO₂:

Z is —H, —C(O)R³, —(C₀₋₁-alkyl)-SO₂—(C₁₋₄-alkyl), —C₁₋₈-alkyl, —CH₂OH,CH₂(O)(C₁₋₈-alkyl) or —CN;

R₁ and R₂ are each independently —CHF₂, —C₁₋₈-alkyl, —C₃₋₁₈-cycloalkyl,or —(C₁₋₁₀-alkyl)(C₃₋₁₈-cycloalkyl), and at least one of R₁ and R₂ isCHF₂;

R³ is —NR⁴R⁵, -alkyl, —OH, —O-alkyl, phenyl, benzyl, substituted phenyl,or substituted benzyl;

R⁴ and R⁵ are each independently —H, —C₁₋₈-alkyl, —OH, —OC(O)R⁶;

R⁶ is —C₁₋₈-alkyl, -amino(C₁₋₈-alkyl), -phenyl, -benzyl, or -aryl;

X₁, X₂, X₃, and X₄ are each independent —H, -halogen, -nitro, —NH₂,—CF₃, —C₁₋₆-alkyl, —(C₀₋₄-alkyl)-(C₃₋₆-cycloalkyl), (C₀₋₄(R⁸),(C₀₋₄-alkyl)-N(H)C(O)N(R⁷R⁸), (C₀₋₄-alkyl)-N(H)C(O)O(R⁷R⁸),(C₀₋₄-alkyl)-OR⁸, (C₀₋₄-alkyl)-imidazolyl, (C₀₋₄-alkyl)-pyrrolyl,(C₀₋₄-alkyl)-oxadiazolyl, or (C₀₋₄-alkyl)-triazolyl, or two of X₁, X₂,X₃, and X₄ may be joined together to form a cycloalkyl orheterocycloalkyl ring, (e.g., X₁ and X₂, X₂ and X₃, X₃ and X₄, X₁ andX₃, X₂ and X₄, or X₁ and X₄ may form a 3, 4, 5, 6, or 7 membered ringwhich may be aromatic, thereby forming a bicyclic system with theisoindolyl ring); and R⁷ and R⁸ are each independently H, C₁₋₉-alkyl,C₃₋₆-cycloalkyl, (C₁₋₆-alkyl)-(C₃₋₆-cycloalkyl), (C₁₋₆-alkyl)-N(R⁷R⁸),(C₁₋₆-alkyl)-OR⁸, phenyl, benzyl, or aryl; or a pharmaceuticallyacceptable salt, solvate, hydrate, stereoisomer, clathrate, or prodrugthereof.

In another embodiment, the invention concerns a method of modulating, inparticular, inhibiting the production or lowering the levels of PDE4 ina mammal or a mammalian cell comprising administering to said mammal aneffective amount of a compound of the invention.

In another embodiment, the invention concerns a method of modulating theproduction or lowering the levels of TNF-α in a mammal or a mammaliancell comprising administering to said mammal an effective amount of acompound of the invention.

In yet another embodiment, the invention concerns a method of modulatingthe production, in particular, inhibiting or lowering the levels of MMPin a mammal or a mammalian cell comprising administering to said mammalan effective amount of a compound of the invention.

Pharmaceutical compositions, modes of administration, formulations, andmethods of using the above compounds alone or in combination aredescribed in more detail below.

3.1 Abbreviations and Definitions

The abbreviations used herein are conventional, unless otherwisedefined.

The terms “treat”, “treating” and “treatment”, as used herein, are meantto include:

(1) alleviating or abrogating a disease and/or its attendant symptoms;

(2) barring a subject from acquiring a disease;

(3) reducing a subject's risk of acquiring a disease;

(4) decreasing the probability or eliminating the possibility that adisease will be contracted;

(5) preventing the disease, i.e., causing the clinical symptoms of thedisease not to develop in a mammal that may be exposed to or predisposedto the disease but does not yet experience or display symptoms of thedisease;

(6) inhibiting the disease, i.e., arresting or reducing the developmentof the disease or its clinical symptoms; or

(7) relieving the disease, i.e., causing regression of the disease orits clinical symptoms.

The term “therapeutically effective amount” refers to that amount of thecompound being administered sufficient to prevent development of oralleviate to some extent one or more of the symptoms of the condition ordisorder being treated as well as to alleviate or eradicate the cause ofthe disease itself.

As used herein, the term “PDE4-responsive condition or disorder” or“mediated by PDE4 inhibition” or “mediated by inhibition of PDE4” refersto a condition or disorder that responds favorably to modulation of PDE4activity. Favorable responses to PDE4 modulation include alleviation orabrogation of the disease and/or its attendant symptoms, inhibition ofthe disease, i.e., arrest or reduction of the development of thedisease, or its clinical symptoms, and regression of the disease or itsclinical symptoms. A PDE4-responsive condition or disease may becompletely or partially responsive to PDE4 modulation. A PDE4-responsivecondition or disorder may be associated with inappropriate, e.g., lessthan or greater than normal, PDE4-activity. Inappropriate PDE4functional activity might arise as the result of PDE4 expression incells which normally do not express PDE4, decreased PDE4 expression(leading to, e.g., lipid and metabolic disorders and diseases) orincreased PDE4 expression. A PDE4-responsive condition or diseaseincludes a PDE4-mediated condition or disease.

The term “alkyl,” by itself or as part of another substituent, means,unless otherwise stated, a straight or branched chain, acyclic or cyclichydrocarbon radical, or combination thereof, which may be fullysaturated, mono- or polyunsaturated and can include di- and multi-valentradicals, having the number of carbon atoms designated (i.e. C₀₋₁₀ meansone to ten carbons, or not present, i.e., when C equals 0, a direct bondexists). Examples of saturated hydrocarbon radicals include groups suchas methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl,sec-butyl, cyclohexyl, (cyclohexyl)methyl, cyclopropylmethyl, homologsand isomers of, for example, n-pentyl, n-hexyl, n-heptyl, n-octyl, andthe like. An unsaturated alkyl group is one having one or more doublebonds or triple bonds. Examples of unsaturated alkyl groups includevinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl),2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl,3-butynyl, and the higher homologs and isomers. The term “alkyl,” unlessotherwise noted, is also meant to include those derivatives of alkyldefined in more detail below as “heteroalkyl,” “cycloalkyl” and“alkylene.” The term “alkylene” by itself or as part of anothersubstituent means a divalent radical derived from an alkane, asexemplified by —CH₂CH₂CH₂CH₂—. Typically, an alkyl group will have from1 to 24 carbon atoms, with those groups having 10 or fewer carbon atomsbeing preferred in the present invention. A “lower alkyl” or “loweralkylene” is a shorter chain alkyl or alkylene group, generally havingeight or fewer carbon atoms.

The term “heteroalkyl,” by itself or in combination with another term,means, unless otherwise stated, a stable straight or branched chain,acyclic or cyclic hydrocarbon radical, or combinations thereof,consisting of the stated number of carbon atoms and from one to threeheteroatoms selected from the group consisting of O, N, Si and S, andwherein the nitrogen and sulfur atoms may optionally be oxidized and thenitrogen heteroatom may optionally be quaternized. The heteroatom(s) O,N and S may be placed at any interior position of the heteroalkyl group.The heteroatom Si may be placed at any position of the heteroalkylgroup, including the position at which the alkyl group is attached tothe remainder of the molecule. Examples include —CH₂—CH₂—O—CH₃,—CH₂—CH₂—NH—CH₃, —CH₂—CH₂—N(CH₃)—CH₃, —CH₂—S—CH₂—CH₃, —CH₂—CH₂—S(O)—CH₃,—CH₂—CH₂—S(O)₂—CH₃, —CH═CH—O—CH₃, —Si(CH₃)₃, —CH₂—CH═N—OCH₃, and—CH═CH—N(CH₃)—CH₃. Up to two heteroatoms may be consecutive, such as,for example, —CH₂—NH—OCH₃ and —CH₂—O—Si(CH₃)₃. Also included in the term“heteroalkyl” are those radicals described in more detail below as“heteroalkylene” and “heterocycloalkyl.” The term “heteroalkylene” byitself or as part of another substituent means a divalent radicalderived from heteroalkyl, as exemplified by —CH₂—CH₂—S—CH₂CH₂— and—CH₂—S—CH₂—CH₂—NH—CH₂—. For heteroalkylene groups, heteroatoms can alsooccupy either or both of the chain termini. Still further, for alkyleneand heteroalkylene linking groups, no orientation of the linking groupis implied.

The terms “cycloalkyl” and “heterocycloalkyl”, by themselves or incombination with other terms, represent, unless otherwise stated, cyclicversions of “alkyl” and “heteroalkyl”, respectively. Thus, the terms“cycloalkyl” and “heterocycloalkyl” are meant to be included in theterms “alkyl” and “heteroalkyl”, respectively. Furthermore, the term“C3-18 cycloalkyl”, means a cycloalkyl with 3 to 18 carbon atoms.Additionally, for heterocycloalkyl, a heteroatom can occupy the positionat which the heterocycle is attached to the remainder of the molecule.Examples of cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like.Examples of heterocycloalkyl include 1-(1,2,5,6-tetrahydropyridyl),1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl,3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl,tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1-piperazinyl,2-piperazinyl, and the like.

The terms “halo” or “halogen,” by themselves or as part of anothersubstituent, mean, unless otherwise stated, a fluorine, chlorine,bromine, or iodine atom. Additionally, terms such as “haloalkyl”, aremeant to include alkyl substituted with halogen atoms which can be thesame or different, in a number ranging from one to (2 m′+1), where m′ isthe total number of carbon atoms in the alkyl group. For example, theterm “halo(C₁-C₄)alkyl” is meant to include trifluoromethyl,2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the like. Thus,the term “haloalkyl” includes monohaloalkyl (alkyl substituted with onehalogen atom) and polyhaloalkyl (alkyl substituted with halogen atoms ina number ranging from two to (2 m′+1) halogen atoms, where m′ is thetotal number of carbon atoms in the alkyl group). The term“perhaloalkyl” means, unless otherwise stated, alkyl substituted with (2m′+1) halogen atoms, where m′ is the total number of carbon atoms in thealkyl group. For example, the term “perhalo(C₁-C₄)alkyl”, is meant toinclude trifluoromethyl, pentachloroethyl,1,1,1-trifluoro-2-bromo-2-chloroethyl, and the like.

The term “aryl,” employed alone or in combination with other terms(e.g., aryloxy, arylthioxy, arylalkyl) means, unless otherwise stated,an aromatic substituent which can be a single ring or multiple rings (upto three rings) which are fused together or linked covalently. The ringsmay each contain from zero to four heteroatoms selected from N, O, andS, wherein the nitrogen and sulfur atoms are optionally oxidized, andthe nitrogen atom(s) are optionally quaternized. The aryl groups thatcontain heteroatoms may be referred to as “heteroaryl” and can beattached to the remainder of the molecule through a heteroatom.Non-limiting examples of aryl groups include phenyl, 1-naphthyl,2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl,2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl,2-phenyl-4-oxazolyl, 5-oxazolyl, oxadiazolyl, 3-isoxazolyl,4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl,2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl,2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl,5-indolyl, triazolyl, 1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl,5-quinoxalinyl, 3-quinolyl, and 6-quinolyl. Substituents for each of theabove noted aryl ring systems are selected from the group of acceptablesubstituents described below.

The term “arylalkyl” is meant to include those radicals in which an arylgroup is attached to an alkyl group (e.g., benzyl, phenethyl,pyridylmethyl and the like) or a heteroalkyl group (e.g., phenoxymethyl,2-pyridyloxymethyl, 341-naphthyloxy)propyl, and the like).

Each of the above terms (e.g., “alkyl,” “heteroalkyl” and “aryl”) ismeant to include both substituted and unsubstituted forms of theindicated radical. Preferred substituents for each type of radical areprovided below.

Substituents for the alkyl and heteroalkyl radicals (including thosegroups often referred to as alkylene, alkenyl, heteroalkylene,heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, andheterocycloalkenyl) can be a variety of groups selected from: —OR′, ═O,═NR′, ═N—OR′, —NR′R″, —SR′, -halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′,—CO₂R′, —CONR′R″, —OC(O)NR′R″, —NR″C(O)R′, —NR′—C(O)NR″R″′, —NR″C(O)₂R′,—NH—C(NH₂)═NH, —NR′C(NH₂)═NH, —NH—C(NH₂)═NR′, —S(O)R′, —S(O)₂R′,—S(O)₂NR′R″, —CN and —NO₂ in a number ranging from zero to (2N+1), whereN is the total number of carbon atoms in such radical. R′, R″ and R″′each independently refer to hydrogen, unsubstituted(C₁-C₈)alkyl andheteroalkyl, unsubstituted aryl, aryl substituted with 1-3 halogens,unsubstituted alkyl, alkoxy or thioalkoxy groups, or aryl-(C₁-C₄)alkylgroups. When R′ and R″ are attached to the same nitrogen atom, they canbe combined with the carbon atoms to which they are attached with thenitrogen atom to form a 5-, 6- or 7-membered ring containing from 1 to 3heteroatoms selected from the group consisting of N, O and S. Forexample, —NR′R″ is meant to include 1-pyrrolidinyl and 4-morpholinyl.From the above discussion of substituents, one of skill in the art willunderstand that the term “alkyl” is meant to include substituted alkylgroups including haloalkyl (e.g., —CF₃ and —CH₂CF₃) and acyl (e.g.,—C(O)CH₃, —C(O)CF₃, —C(O)CH₂OCH₃, and the like).

Similarly, substituents for the aryl groups are varied and are selectedfrom: -halogen, —OR′, —OC(O)R′, —NR′R″, —SR′, —R′, —CN, —NO₂, —CO₂R′,—CONR′R″, —C(O)R′, —OC(O)NR′R″, —NR″C(O)R′, —NR″C(O)₂R′, C(O)NR″R′″,—NH—C(NH₂)═NH, —NR′C(NH₂)═NH, —NH—C(NH₂)—NR′, —S(O)R′, —S(O)₂R′,—S(O)₂NR′R″, —N₃, —CH(Ph)₂, fluoro(C₁-C₄)alkoxy, and fluoro(C₁-C₄)alkyl,in a number ranging from zero to the total number of open valences onthe aromatic ring system; and where each R′, R″ and R′ is independentlyselected from hydrogen, (C₁-C₈)alkyl and heteroalkyl, unsubstitutedaryl, (unsubstituted aryl)-(C₁-C₄)alkyl and (unsubstitutedaryl)oxy-(C₁-C₄)alkyl.

As used herein, the term “heteroatom” is meant to include oxygen (O),nitrogen (N), sulfur (S) and silicon (Si).

The term “pharmaceutically acceptable salts” is meant to include saltsof the active compounds which are prepared with relatively nontoxicacids or bases, depending on the particular substituents found on thecompounds described herein. When compounds of the present inventioncontain relatively acidic functionalities, base addition salts can beobtained by contacting the neutral form of such compounds with asufficient amount of the desired base, either neat or in a suitableinert solvent. Examples of pharmaceutically acceptable base additionsalts include sodium, potassium, calcium, ammonium, organic amino, ormagnesium salt, or a similar salt. When compounds of the presentinvention contain relatively basic functionalities, acid addition saltscan be obtained by contacting the neutral form of such compounds with asufficient amount of the desired acid, either neat or in a suitableinert solvent. Examples of pharmaceutically acceptable acid additionsalts include those derived from inorganic acids like hydrochloric,hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric,monohydrogenphosphoric, dihydrogenphosphoric, sulfuric,monohydrogensulfuric, hydriodic, or phosphorous acids and the like, aswell as the salts derived from relatively nontoxic organic acids likeacetic, propionic, isobutyric, oxalic, maleic, malonic, benzoic,succinic, suberic, fumaric, mandelic, phthalic, benzenesulfonic,p-tolylsulfonic, citric, tartaric, methanesulfonic, and the like. Alsoincluded are salts of amino acids such as arginate and the like, andsalts of organic acids like glucuronic or galactunoric acids and thelike (see, for example, Berge et al. (1977) J. Pharm. Sci. 66:1-19).Certain specific compounds of the present invention contain both basicand acidic functionalities that allow the compounds to be converted intoeither base or acid addition salts.

The neutral forms of the compounds may be regenerated by contacting thesalt with a base or acid and isolating the parent compound in theconventional manner. The parent form of the compound differs from thevarious salt forms in certain physical properties, such as solubility inpolar solvents, but otherwise the salts are equivalent to the parentform of the compound for the purposes of the present invention.

In addition to salt forms, the present invention provides compoundswhich are in a prodrug form. As used herein and unless otherwiseindicated, the term “prodrug” means a derivative of a compound that canhydrolyze, oxidize, or otherwise react under biological conditions (invitro or in vivo) to provide the compound. Examples of prodrugs include,but are not limited to, derivatives of compounds of the invention thatcomprise biohydrolyzable moieties such as biohydrolyzable amides,biohydrolyzable esters, biohydrolyzable carbamates, biohydrolyzablecarbonates, biohydrolyzable ureides, and biohydrolyzable phosphateanalogues. Other examples of prodrugs include derivatives of compoundsof the invention that comprise —NO, —NO₂, —ONO, or —ONO₂ moieties.Prodrugs can typically be prepared using well-known methods, such asthose described in 1 Burger's Medicinal Chemistry and Drug Discovery,172-178, 949-982 (Manfred E. Wolff ed., 5th ed. 1995), and Design ofProdrugs(H. Bundgaard ed., Elselvier, N.Y. 1985).

As used herein and unless otherwise indicated, the terms“biohydrolyzable amide,” “biohydrolyzable ester,” “biohydrolyzablecarbamate,” “biohydrolyzable carbonate,” “biohydrolyzable ureide,”“biohydrolyzable phosphate” mean an amide, ester, carbamate, carbonate,ureide, or phosphate, respectively, of a compound that either: 1) doesnot interfere with the biological activity of the compound but canconfer upon that compound advantageous properties in vivo, such asuptake, duration of action, or onset of action; or 2) is biologicallyinactive but is converted in vivo to the biologically active compound.Examples of biohydrolyzable esters include, but are not limited to,lower alkyl esters, lower acyloxyalkyl esters (such as acetoxylmethyl,acetoxyethyl, aminocarbonyloxymethyl, pivaloyloxymethyl, andpivaloyloxyethyl esters), lactonyl esters (such as phthalidyl andthiophthalidyl esters), lower alkoxyacyloxyalkyl esters (such asmethoxycarbonyloxymethyl, ethoxycarbonyloxyethyl andisopropoxycarbonyloxyethyl esters), alkoxyalkyl esters, choline esters,and acylamino alkyl esters (such as acetamidomethyl esters). Examples ofbiohydrolyzable amides include, but are not limited to, lower alkylamides, ∀-amino acid amides, alkoxyacyl amides, andalkylaminoalkylcarbonyl amides. Examples of biohydrolyzable carbamatesinclude, but are not limited to, lower alkylamines, substitutedethylenediamines, aminoacids, hydroxyalkylamines, heterocyclic andheteroaromatic amines, and polyether amines.

Certain compounds of the present invention can exist in unsolvated formsas well as solvated forms, including hydrated forms. In general, thesolvated forms are equivalent to unsolvated forms and are intended to beencompassed within the scope of the present invention. Certain compoundsof the present invention may exist in multiple crystalline or amorphousforms. In general, all physical forms are equivalent for the usescontemplated by the present invention and are intended to be within thescope of the present invention.

Certain compounds of the present invention possess asymmetric carbonatoms (optical centers) or double bonds; the racemates, enantiomers,diastereomers, geometric isomers and individual isomers are all intendedto be encompassed within the scope of the present invention. Theseisomers can be resolved or asymmetrically synthesized using conventionalmethods to render the isomers “optically pure” i.e., substantially freeof its other isomers; preferably, 85%, 90%, 95% or 97% ee. Preferably,the compounds of the invention are administered as substantially pure(R) or (S) enantiomers, substantially free of its opposite enantiomer.

The compounds of the present invention may also contain unnaturalproportions of atomic isotopes at one or more of the atoms thatconstitute such compounds. For example, the compounds may beradiolabeled with radioactive isotopes, such as for example tritium(³H), iodine-125 (¹²⁵I) or carbon-14 (¹⁴C). Radiolabeled compounds areuseful as therapeutic agents, e.g., cancer therapeutic agents, researchreagents, e.g., assay reagents, and diagnostic agents, e.g., in vivoimaging agents. All isotopic variations of the compounds of the presentinvention, whether radioactive or not, are intended to be encompassedwithin the scope of the present invention.

4. DETAILED DESCRIPTION

The invention encompasses novel compounds and compositions thereof thatare useful to treat or prevent diseases in mammals, including humans.The invention further encompasses the use of these compounds fortreating or preventing diseases or disorders including, but not limitedto, cancer; viral, genetic, inflammatory, allergic, and autoimmunedisease; and bacterial infections. The compounds of the invention areparticularly useful to treat or prevent diseases caused or aggravated byexcessive, insufficient or unregulated levels of PDE4, TNF-α, or MMP.

The compounds provided herein have the formula (I):

wherein:

Y is —C(O)—, —CH₂, —CH₂C(O)—, —C(O)CH₂—, or SO₂,

Z is —H, —C(O)R³, —C₁₋₈-alkyl, —CH₂OH, CH₂(O)(C₁₋₈-alkyl) or —CN;

R₁ and R₂ are each independently —CHF₂, —C₁₋₈-alkyl, —C₃₋₁₈-cycloalkyl,or —(C₁₋₁₀-alkyl)(C₃₋₁₈-cycloalkyl), and at least one of R₁ and R₂ isCHF₂;

R³ is —NR⁴R⁵, -alkyl, —OH, —O-alkyl, phenyl, benzyl, substituted phenyl,or substituted benzyl;

R⁴ and R⁵ are each independently —H, —C₁₋₈-alkyl, —OH, —OC(O)R⁶;

R⁶ is —C₁₋₈-alkyl, -amino(C₁₋₈-alkyl), -phenyl, -benzyl, or -aryl;

X₁, X₂, X₃, and X₄ are each independent —H, -halogen, -nitro, —NH₂,—CF₃, —C₁₋₆-alkyl, —(C₀₋₄-alkyl)-(C₃₋₆-cycloalkyl), (C₀₋₄-alkyl)-NR⁷R⁸,(C₀₋₄-alkyl)-N(H)C(O)—(R⁸), (C₀₋₄-alkyl)-N(H)C(O)N(R⁷R⁸),(C₀₋₄-alkyl)-N(H)C(O)O(R⁷R⁸), (C₀₋₄-alkyl)-OR⁸, (C₀₋₄-alkyl)-imidazolyl,(C₀₋₄-alkyl)-pyrrolyl, (C₀₋₄-alkyl)-oxadiazolyl, or(C₀₋₄-alkyl)-triazolyl, or two of X₁, X₂, X₃, and X₄ may be joinedtogether to form a cycloalkyl or heterocycloalkyl ring, (e.g., X₁ andX₂, X₂ and X₃, X₃ and X₄, X₁ and X₃, X₂ and X₄, or X₁ and X₄ may form a3, 4, 5, 6, or 7 membered ring which may be aromatic, thereby forming abicyclic system with the isoindolyl ring); and

R⁷ and R⁸ are each independently H, C₁₋₆-cycloalkyl,(C₁₋₆-alkyl)-(C₃₋₆-cycloalkyl), (C₁₋₆-alkyl)-N(R⁷R⁸), (C₁₋₆-alkyl)-OR⁸,phenyl, benzyl, or aryl; or a pharmaceutically acceptable salt, solvate,hydrate, stereoisomer, clathrate, or prodrug thereof.

In a preferred embodiment, at least one of X₁, X₂, X₃, and X₄ is(C₀₋₄-alkyl)-N—(R⁷R⁸)₂.

In another preferred embodiment, at least one of X₁, X₂, X₃, and X₄ is(C₀₋₄-alkyl)-NHC═O—(R⁸).

In another preferred embodiment, at least one of X₁, X₂, X₃, and X₄ is(C₀₋₄-alkyl)-NHC═ON(R⁷R⁸).

In another preferred embodiment, at least one of X₁, X₂, X₃, and X₄ is(C₀₋₄-alkyl)-NHC═OO(R⁷R⁸).

In another preferred embodiment, at least one of X₁, X₂, X₃, and X₄ is(C₀₋₄-alkyl)-O—R⁸

In another preferred embodiment, at least one of X₁, X₂, X₃, and X₄ isNH₂.

In another preferred embodiment, at least one of X_(i), X₂, X₃, and X₄is (C₀₋₄-alkyl)-NHC(O)(R⁸).

In another preferred embodiment, at least one of X₁, X₂, X₃, and X₄ is-halogen.

In another preferred embodiment, at least one of X₁, X₂, X₃, and X₄ is(C₀₋₄-alkyl)-imidazolyl, (C₀₋₄-alkyl)-pyrrolyl,(C₀₋₄-alkyl)-oxadiazolyl, or (C₀₋₄-alkyl)-triazolyl.

In another preferred embodiment, at least one of X₁, X₂, X₃, and X₄ is(C₀₋₄-alkyl)-cyclopropyl.

In another preferred embodiment, at least one of X₁, X₂, X₃, and X₄ is(C₀₋₄-alkyl)-NHC(O)(R⁸) and one of X₁, X₂, X₃, and X₄ is halogen.

In still another preferred embodiment, three of X₁, X₂, X₃, and X₄ areH.

In an alternative embodiment, either X₁ or X₂ is substituted.

In a preferred embodiment, R₁ or R₂ is a —CHF₂ and the other is a C₁₋₄alkyl or a C₃₋₆ cycloalkyl. More preferably, R₁ or R₂ is independently,methyl, ethyl, cyclopentyl or —CHF₂. Most preferably, one of R₁ or R₂must be —CHF₂.

In another preferred embodiment, Y is —C(O)— or CH₂.

In another preferred embodiment Z is —C(O)R³, —(C₀₋₁-alkyl)-SO₂-alkyl,—CH₂OH, —CHNH₂, —SO₂CH₃, —C(O)CH₃, —C(O)NHOH or —CH₂N(CH₃)₂.

In an alternative embodiment, Z is a hydroxyalkyl group of 1 to 6 carbonatoms.

The compounds of the invention generally exist in solid form and can berecrystallized according to well-known methods affording high-puritycrystals, preferably, in greater than 95% purity, more preferably, ingreater than 98% purity. Narrow melting-point range is an indication ofpurity, thus, compounds of the invention generally have a melting pointwithin a range of 3° C. to 4° C., more preferably, within a range of 2°C.

Various compounds of the invention contain one or more chiral centers,and can exist as racemic mixtures of enantiomers or mixtures ofdiastereomers. This invention encompasses the use of stereomericallypure forms of such compounds, as well as the use of mixtures of thoseforms. For example, mixtures comprising equal or unequal amounts of theenantiomers of a particular compounds of the invention may be used inmethods and compositions of the invention. These isomers may beasymmetrically synthesized or resolved using standard techniques such aschiral columns or chiral resolving agents. See, e.g., Jacques, J., etal., Enantiomers, Racemates and Resolutions (Wiley-Interscience, NewYork, 1981); Wilen, S. H., et al., Tetrahedron 33:2725 (1977); Eliel, E.L., Stereochemistry of Carbon Compounds (McGraw-Hill, N.Y., 1962); andWilen, S. H., Tables of Resolving Agents and Optical Resolutions p. 268(E. L. Eliel, Ed., Univ. of Notre Dame Press, Notre Dame, 1N, 1972).

The compounds of the invention can contain one or more chiral centersand/or double bonds and, therefore, exist as stereoisomers, such asdouble-bond isomers (i.e., geometric isomers), enantiomers, ordiastereomers. According to the invention, the chemical structuresdepicted herein, and therefore the compounds of the invention, encompassall of the corresponding enantiomers and stereoisomers, that is, boththe stereomerically pure form (e.g., geometrically pure,enantiomerically pure, or diastereomerically pure) and enantiomeric andstereoisomeric mixtures, e.g., racemates.

A compound of the invention is considered optically active orenantiomerically pure (i.e., substantially the R-form or substantiallythe S-form) with respect to a chiral center when the compound is about80% ee (enantiomeric excess) or greater, preferably, equal to or greaterthan 90% ee with respect to a particular chiral center, and morepreferably 95% ee with respect to a particular chiral center. Thus, theinvention encompasses all enantiomerically pure, enantiomericallyenriched, and racemic mixtures of compounds of Formulas I.

Enantiomeric and stereoisomeric mixtures of compounds of the inventioncan be resolved into their component enantiomers or stereoisomers bywell-known methods, such as chiral-phase gas chromatography,chiral-phase high performance liquid chromatography, crystallizing thecompound as a chiral salt complex, or crystallizing the compound in achiral solvent. Enantiomers and stereoisomers can also be obtained fromstereomerically or enantiomerically pure intermediates, reagents, andcatalysts by well-known asymmetric synthetic methods.

The invention further encompasses prodrugs of compounds falling withinFormula I. The term “prodrug” refers to a compound that, followingadministration in a mammal, converts, via a biotransformation, into acompound falling within Formula I in vivo. Prodrugs of compounds fallingwithin Formula I can be synthesized using well-known methods, such asthose described by Burger's Medicinal Chemistry and Drug Chemistry,Fifth Ed., Vol. 1, pp. 172-178, 949-982 (1995).

The compounds of the invention are defined herein by their chemicalstructures and/or chemical names. Where a compound is referred to byboth a chemical structure and a chemical name, and the chemicalstructure and chemical name conflict, the chemical structure isdeterminative of the compound's identity.

In another embodiment, the present invention further providespharmaceutical compositions comprising a therapeutically effective or aprophylactically effective amount of one or more compounds of theinvention and a pharmaceutically acceptable vehicle or carrier. Apharmaceutically acceptable vehicle or carrier can comprise anexcipient, diluent, or a mixture thereof. The term “therapeuticallyeffective amount” means the amount of a compound of the invention thatwill elicit the biological or medical response in a mammal that is beingthat is being treated by the veterinarian or clinician. The term“prophylactically effective” means the amount of a compound of theinvention that will prevent or inhibit affliction or mitigate afflictionof a mammal with a medical condition that a veterinarian or clinician istrying to prevent, inhibit, or mitigate.

In another embodiment, the invention concerns a method inhibiting PDE4in a mammal comprising administering to said mammal an effective amountof a compound of the invention.

In another embodiment, the invention concerns a method of modulating theproduction or lowering the levels of TNF-α in a mammal comprisingadministering to said mammal an effective amount of a compound of theinvention.

In yet another embodiment, the invention concerns a method of inhibitingMMP in a mammal comprising administering to said mammal an effectiveamount of a compound of the invention.

In yet another embodiment, the invention concerns a method of treatingundesired angiogenesis in a mammal comprising administering to saidmammal an effective amount of a compound of the invention. Diseasesassociated with angiogenesis are well known in the art.

A separate embodiment of the invention encompasses methods of treatingor preventing Myelodysplastic syndrome (MDS) which comprisesadministering to a patient in need of such treatment or prevention atherapeutically or prophylactically effective amount of a compound ofthe invention, or a pharmaceutically acceptable salt, solvate, hydrate,stereoisomer, clathrate, or prodrug thereof. MDS refers to a diversegroup of hematopoietic stem cell disorders. MDS is characterized by acellular marrow with impaired morphology and maturation(dysmyelopoiesis), peripheral blood cytopenias, and a variable risk ofprogression to acute leukemia, resulting from ineffective blood cellproduction. The Merck Manual 953 (17th ed. 1999) and List et al., 1990,J. Clin. Oncol. 8:1424.

A separate embodiment of the invention encompasses methods of treatingor preventing Myeloproliferative disease (MPD) which comprisesadministering to a patient in need of such treatment or prevention atherapeutically or prophylactically effective amount of a compound ofthe invention, or a pharmaceutically acceptable salt, solvate, hydrate,stereoisomer, clathrate, or prodrug thereof. Myeloproliferative disease(MPD) refers to a group of disorders characterized by clonalabnormalities of the hematopoietic stem cell. See e.g., Current MedicalDiagnosis & Treatment, pp. 499 (37th ed., Tierney et al. ed, Appleton &Lange, 1998).

The invention also encompasses a method of treating, preventing ormanaging complex regional pain syndrome, which comprises administeringto a patient in need of such treatment, prevention or management atherapeutically or prophylactically effective amount of a compound ofthe invention, or a pharmaceutically acceptable salt, solvate, hydrate,stereoisomer, clathrate, or prodrug thereof, before, during or aftersurgery or physical therapy directed at reducing or avoiding a symptomof complex regional pain syndrome in the patient.

In still another embodiment, the invention concerns a method of treatingor preventing cancer in a mammal, comprising administering to saidmammal a therapeutically effective amount of a compound of theinvention. The compounds of the invention can be used to treat orprevent any cancer, for example, solid tumors and blood-born tumors.Specific examples of cancers treatable or preventable by compounds ofthe invention include, but are not limited to, cancers of the skin, suchas melanoma; lymph node; breast; cervix; uterus; gastrointestinal tract;lung; ovary; prostate; colon; rectum; mouth; brain; head and neck;throat; testes; kidney; pancreas; bone; spleen; liver; bladder; larynx;nasal passages; and AIDS-related cancers. The compounds are particularlyuseful for treating cancers of the blood and bone marrow, such asmultiple myeloma and acute and chronic leukemias, for example,lymphoblastic, myelogenous, lymphocytic, and myelocytic leukemias. Thecompounds of the invention can be used for treating or preventing eitherprimary or metastatic tumors.

In yet one more embodiment, the invention provides methods of treatingor preventing cancer in a mammal, comprising administering to a mammalin need thereof, a therapeutically effective amount of a compound of theinvention and another therapeutic agent.

In yet another embodiment, the invention concerns a method of treatingor preventing inflammatory disorders in a mammal, comprisingadministering to said mammal a therapeutically effective amount of acompound of the invention. The compounds of the invention are especiallyeffective to treat or prevent inflammatory diseases related to theup-regulation of TNF-α including, but not limited to, arthriticconditions, such as, rheumatoid arthritis, and osteoarthritis;rheumatoid spondylitis; psoriasis; post ischemic perfusion injury;inflammatory bowel disease; and chronic inflammatory pulmonary disease.

In one more embodiment still, the invention provides methods of treatingor preventing inflammatory disorders in a mammal, comprisingadministering to a mammal in need thereof, a therapeutically effectiveamount of a compound of the invention and another anti-inflammatoryagent.

In a further embodiment, the invention concerns a method of treating orpreventing heart disease in a mammal comprising administering to saidmammal a therapeutically effective amount of a compound of theinvention. For example, the compounds of the invention can be used totreat or prevent congestive heart failure, cardiomyopathy, pulmonaryedema, endotoxin-mediated septic shock, acute viral myocarditis, cardiacallograft rejection, and myocardial infarction.

In an additional embodiment, the invention concerns a method of treatingor preventing osteoporosis in a mammal comprising administering to saidmammal a therapeutically effective amount of a compound of theinvention.

In a further embodiment, the invention relates to a method of treatingor preventing viral, genetic, allergic, and autoimmune diseases. Forexample, the compounds are useful to treat or prevent diseasesincluding, but not limited to, HIV, hepatitis, adult respiratorydistress syndrome, bone resorption diseases, chronic pulmonaryinflammatory diseases, dermatitis, cystic fibrosis, septic shock,sepsis, endotoxic shock, hemodynamic shock, sepsis syndrome, postischemic reperfusion injury, meningitis, psoriasis, fibrotic disease,cachexia, graft versus host disease, graft rejection, auto-immunedisease, rheumatoid spondylitis, Crohn's disease, ulcerative colitis,inflammatory-bowel disease, multiple sclerosis, systemic lupuserythrematosus, ENL in leprosy, radiation damage, cancer, asthma, orhyperoxic alveolar injury in a mammal comprising administering to saidmammal a therapeutically effective amount of a compound of theinvention.

In still another embodiment, the invention concerns a method of treatingor preventing malaria, mycobacterial infection, or an opportunisticinfection resulting from HIV in a mammal, comprising administering tosaid mammal a therapeutically effective amount of a compound of theinvention.

In still one more embodiment, the invention relates to treating orpreventing mammals having more than one of the conditions treatable by acompound of the invention.

In the above embodiments, it is preferable that the mammal be in need ofthe treatment or prevention, that is, the mammal is actually sufferingfrom a medical condition or at risk of a medical condition for which acompound of the invention can provide treatment or prevention. However,the compounds of the invention can also be administered to test animalsthat do not necessarily require such treatment or prevention.

In a further embodiment, the invention encompasses a method ofmodulating the production, preferably inhibiting, or lowering the levelsof PDE4 in a mammalian cell or tissue comprising contacting an effectiveamount of a compound of the invention with said mammalian cell ortissue.

In a further embodiment, the invention encompasses a method ofmodulating the production or lowering the levels of TNF-α in a mammaliancell or tissue comprising contacting an effective amount of a compoundof the invention with said mammalian cell or tissue.

In yet another embodiment, the invention encompasses a method ofmodulating the production or lowering the levels of MMP in a mammaliancell or tissue comprising contacting an effective amount of a compoundof the invention with said mammalian cell or tissue.

In these embodiments, the term “effective amount” means the amount ofthe compound that will induce the biological response sought by theresearcher, veterinarian, physician, or clinician. It should beunderstood that the cell can be in a cell culture or a tissue culture(in vitro) or in an organism (in vivo) including a human.

The present invention may be understood by reference to the detaileddescription and examples that are intended to exemplify non-limitingembodiments of the invention.

4.1 Preparation of the Compounds

The compounds can be prepared using methods which are known in generalfor the preparation of imides and 2,3-dihydro-1H-isoindolinones.However, the present invention also pertains to an improvement in theformation of the final compounds, as discussed below in greater detail.

An N-alkoxycarbonylimide and an amine thus are allowed to react in thepresence of a base such as sodium carbonate or sodium bicarbonatesubstantially as described by Shealy et al., Chem. & Ind., (1965)1030-1031) and Shealy et al., J. Pharm. Sci. 57, 757-764 (1968) to yieldthe N-substituted imide. Alternatively, a cyclic acid anhydride can bereacted with an appropriate amine to form an imide. Formation of acyclic imide also can be accomplished by refluxing a solution of anappropriately substituted dicarboxylic acid monoamide in anhydroustetrahydrofuran with N,N′-carbonyldiimidazole. Also, a2-bromomethyl-benzoic ester can be reacted with an appropriate amine toform a 2,3-dihydro-1H-isoindolinone as shown below.

Other methods of formation are described in U.S. Pat. No. 5,605,914 andInternational Publication No. WO 01/34606 A1 which are incorporatedherein in there entireties by reference.

4.2 Pharmaceutical Compositions

In another aspect, the present invention provides pharmaceuticalcompositions comprising a pharmaceutically acceptable carrier, excipientor diluent and one or more compounds of the present invention.

One embodiment provides the subject compounds combined with apharmaceutically acceptable excipient such as sterile saline,methylcellulose solutions, detergent solutions or other medium, water,gelatin, oils, etc. The compounds or compositions may be administeredalone or in combination with any convenient carrier, diluent, etc., andsuch administration may be provided in single or multiple dosages. Thecompositions are sterile, particularly when used for parenteraldelivery. However, oral unit dosage forms need not be sterile. Usefulcarriers include water soluble and water insoluble solids, fatty acids,micelles, inverse micelles, liposomes and semi-solid or liquid media,including aqueous solutions and non-toxic organic solvents. All of theabove formulations may be treated with ultrasounds, stirred, mixed,high-shear mixed, heated, ground, milled, aerosolized, pulverized,lyophilized, etc., to form pharmaceutically acceptable compositions.

For preparing pharmaceutical compositions from the compounds of thepresent invention, pharmaceutically acceptable carriers can be eithersolid or liquid. Solid form preparations include powders, tablets,pills, capsules, cachets, suppositories, and dispersible granules. Asolid carrier can be one or more substances which may also act asdiluents, flavoring agents, binders, preservatives, tabletdisintegrating agents, or an encapsulating material.

In powders, the carrier is a finely divided solid, which is in a mixturewith the finely divided active component. In tablets, the activecomponent is mixed with the carrier having the necessary bindingproperties in suitable proportions and compacted in the shape and sizedesired.

The powders and tablets preferably contain from 5% or 10% to 70% of theactive compound. Suitable carriers are magnesium carbonate, magnesiumstearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin,tragacanth, methylcellulose, sodium carboxymethylcellulose, a lowmelting wax, cocoa butter, and the like. The term “preparation” isintended to include the formulation of the active compound withencapsulating material as a carrier providing a capsule in which theactive component with or without other carriers, is surrounded by acarrier, which is thus in association with it. Similarly, cachets andlozenges are included. Tablets, powders, capsules, pills, cachets, andlozenges can be used as solid dosage forms suitable for oraladministration.

For preparing suppositories, a low melting wax, such as a mixture offatty acid glycerides or cocoa butter, is first melted and the activecomponent is dispersed homogeneously therein, as by stirring. The moltenhomogeneous mixture is then poured into convenient sized molds, allowedto cool, and thereby to solidify.

Liquid form preparations include solutions, suspensions, and emulsions,for example, water or water/propylene glycol solutions. For parenteralinjection, liquid preparations can be formulated in solution in aqueouspolyethylene glycol solution.

Aqueous solutions suitable for oral use can be prepared by dissolvingthe active component in water and adding suitable colorants, flavors,stabilizers, and thickening agents as desired. Aqueous suspensionssuitable for oral use can be made by dispersing the finely dividedactive component in water with viscous material, such as natural orsynthetic gums, resins, methylcellulose, sodium carboxymethylcellulose,and other well-known suspending agents.

Also included are solid form preparations which are intended to beconverted, shortly before use, to liquid form preparations for oraladministration. Such liquid forms include solutions, suspensions, andemulsions. These preparations may contain, in addition to the activecomponent, colorants, flavors, stabilizers, buffers, artificial andnatural sweeteners, dispersants, thickeners, solubilizing agents, andthe like.

The pharmaceutical preparation is preferably in unit dosage form. Insuch form the preparation is subdivided into unit doses containingappropriate quantities of the active component. The unit dosage form canbe a packaged preparation, the package containing discrete quantities ofpreparation, such as packeted tablets, capsules, and powders in vials orampoules. Also, the unit dosage form can be a capsule, tablet, cachet,or lozenge itself, or it can be the appropriate number of any of thesein packaged form.

The quantity of active component in a unit dose preparation may bevaried or adjusted from 0.1 mg to 1000 mg, preferably 1.0 mg to 100 mgaccording to the particular application and the potency of the activecomponent. The composition can, if desired, also contain othercompatible therapeutic agents.

The pharmaceutical compositions and methods of the present invention mayfurther comprise other therapeutically active compounds, as notedherein, useful in the treatment of metabolic disorders, cardiovasculardiseases, inflammatory conditions or neoplastic diseases and pathologiesassociated therewith (e.g., diabetic neuropathy) or other adjuvant. Inmany instances, compositions which include a compound of the inventionand an alternative agent have additive or synergistic effects whenadministered.

4.3 Methods of Use

In accordance with the invention, a compound or composition of theinvention is administered to a mammal, preferably, a human, with or atrisk of a disease or medical condition, for example, cancer, such assolid tumors and blood-born tumors. Specific examples of cancerstreatable or preventable by administering compounds of the inventioninclude, but are not limited to, cancers of the skin, such as melanoma;lymph node; breast; cervix; uterus; gastrointestinal tract; lung; ovary;prostate; colon; rectal; mouth; brain; head and neck; throat; testes;kidney; pancreas; bone; spleen; liver; bladder; larynx; nasal passages;and AIDS-related cancers. The compounds are particularly useful fortreating cancers of the blood and bone marrow, such as multiple myelomaand acute and chronic leukemias, for example, lymphoblastic,myelogenous, lymphocytic, and myelocytic leukemias.

Other examples of specific cancers that can be treated, prevented ormanaged by methods of this invention include, but are not limited to,advanced malignancy, amyloidosis, neuroblastoma, meningioma,hemangiopericytoma, multiple brain metastases, glioblastoma multiforms,glioblastoma, brain stem glioma, poor prognosis malignant brain tumor,malignant glioma, anaplastic astrocytoma, anaplastic oligodendroglioma,neuroendocrine tumor, rectal adenocarcinoma, Dukes C & D colorectalcancer, unresectable colorectal carcinoma, metastatic hepatocellularcarcinoma, Kaposi's sarcoma, karotype acute myeloblastic leukemia,Hodgkin's lymphoma, non-Hodgkin's lymphoma, cutaneous T-Cell lymphoma,non-cutaneous T-Cell lymphoma, cutaneous B-Cell lymphoma, diffuse largeB-Cell lymphoma, low grade follicular lymphoma, malignant melanoma,malignant mesothelioma, malignant pleural effusion mesotheliomasyndrome, mutiple myeloma, peritoneal carcinoma, papillary serouscarcinoma, gynecologic sarcoma, soft tissue sarcoma, scelroderma, andcutaneous vasculitis.

The compounds of the invention are also useful to treat or prevent heartdisease, such as congestive heart failure, cardiomyopathy, pulmonaryedema, endotoxin-mediated septic shock, acute viral myocarditis, cardiacallograft rejection, and myocardial infarction.

The compounds of the invention can also be used to treat or preventviral, genetic, inflammatory, allergic, and autoimmune diseases. Forexample, the compounds are useful to treat or prevent diseasesincluding, but not limited to, HIV; hepatitis; adult respiratorydistress syndrome; bone-resorption diseases; chronic obstructivepulmonary disease, chronic pulmonary inflammatory diseases; dermatitis;cystic fibrosis; septic shock; sepsis; endotoxic shock; hemodynamicshock; sepsis syndrome; post ischemic reperfusion injury; meningitis;psoriasis; fibrotic disease; cachexia; graft rejection; auto-immunedisease; rheumatoid spondylitis; arthritic conditions, such asrheumatoid arthritis and osteoarthritis; osteoporosis, Crohn's disease;ulcerative colitis; inflammatory-bowel disease; multiple sclerosis;systemic lupus erythrematosus; ENL in leprosy; radiation damage; asthma;and hyperoxic alveolar injury.

The compounds of the invention are also useful for treating orpreventing bacterial infections including, but not limited to, malaria,mycobacterial infection, and opportunistic infections resulting fromHIV.

Another embodiment of the invention encompasses methods of treating,managing or preventing diseases and disorders associated with, orcharacterized by, undesired angiogenesis, which comprise administeringto a patient in need of such treatment, management or prevention atherapeutically or prophylactically effective amount of animmunomodulatory compound of the invention, or a pharmaceuticallyacceptable salt, solvate, hydrate, stereoisomer, clathrate, or prodrugthereof.

Examples of diseases and disorders associated with, or characterized by,undesired angiogenesis include, but are not limited to, inflammatorydiseases, autoimmune diseases, viral diseases, genetic diseases,allergic diseases, bacterial diseases, ocular neovascular diseases,choroidal neovascular diseases, retina neovascular diseases, andrubeosis (neovascularization of the angle), which are mediated byundesired or uncontrolled angiogenesis. In certain embodiment of theinvention, specific diseases do not include congestive heart failure,cardiomyopathy, pulmonary edema, endotoxin-mediated septic shock, acuteviral myocarditis, cardiac allograft rejection, myocardial infarction,HIV, hepatitis, adult respiratory distress syndrome, bone-resorptiondisease, chronic obstructive pulmonary diseases, chronic pulmonaryinflammatory disease, dermatitis, cystic fibrosis, septic shock, sepsis,endotoxic shock, hemodynamic shock, sepsis syndrome, post ischemicreperfusion injury, meningitis, psoriasis, fibrotic disease, cachexia,graft rejection, rheumatoid spondylitis, osteoporosis, Crohn's disease,ulcerative colitis, inflammatory-bowel disease, multiple sclerosis,systemic lupus erythrematosus, erythema nodosum leprosum in leprosy,radiation damage, asthma, hyperoxic alveolar injury, malaria andmycobacterial infection.

The compounds of the invention are also useful for preventing heartdisease, such as congestive heart failure, cardiomyopathy, pulmonaryedema, endotoxin-mediated septic shock, acute viral myocarditis, cardiacallograft rejection, and myocardial infarction.

4.4 Therapeutic/Prophylactic Administration of The Compounds andCompositions of the Invention

Administration of compounds of the invention can be systemic or local.In most instances, administration to a mammal will result in systemicrelease of the compounds of the invention (i.e., into the bloodstream).Methods of administration include enteral routes, such as oral, buccal,sublingual, and rectal; topical administration, such as transdermal andintradermal; and parenteral administration. Suitable parenteral routesinclude injection via a hypodermic needle or catheter, for example,intravenous, intramuscular, subcutaneous, intradermal, intraperitoneal,intraarterial, intraventricular, intrathecal, and intracameral injectionand non-injection routes, such as intravaginal rectal, or nasaladministration. Preferably, the compounds and compositions of theinvention are administered orally. In specific embodiments, it may bedesirable to administer one or more compounds of the invention locallyto the area in need of treatment. This may be achieved, for example, bylocal infusion during surgery, topical application, e.g., in conjunctionwith a wound dressing after surgery, by injection, by means of acatheter, by means of a suppository, or by means of an implant, saidimplant being of a porous, non-porous, or gelatinous material, includingmembranes, such as sialastic membranes, or fibers.

The compounds of the invention can be administered via typical as wellas non-standard delivery systems, e.g., encapsulation in liposomes,microparticles, microcapsules, capsules, etc. For example, the compoundsand compositions of the invention can be delivered in a vesicle, inparticular a liposome (see Langer, 1990, Science 249:1527-1533; Treat etal., in Liposomes in Therapy of Infectious Disease and Cancer,Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989);Lopez-Berestein, ibid., pp. 317-327; see generally ibid.). In anotherexample, the compounds and compositions of the invention can bedelivered in a controlled release system. In one embodiment, a pump maybe used (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng.14:201; Buchwald et al., 1980, Surgery 88:507 Saudek et al., 1989, N.Engl. J. Med. 321:574). In another example, polymeric materials can beused (see Medical Applications of Controlled Release, Langer and Wise(eds.), CRC Press., Boca Raton, Fla. (1974); Controlled DrugBioavailability, Drug Product Design and Performance, Smolen and Ball(eds.), Wiley, New York (1984); Ranger and Peppas, 1983, J. Macromol.Sci. Rev. Macromol. Chem. 23:61; see also Levy et al., 1985, Science228:190; During et al., 1989, Ann. Neurol. 25:351; Howard et al., 1989,J. Neurosurg. 71:105). In still another example, a controlled-releasesystem can be placed in proximity of the target area to be treated,e.g., the liver, thus requiring only a fraction of the systemic dose(see, e.g., Goodson, in Medical Applications of Controlled Release,supra, vol. 2, pp. 115-138 (1984)). Other controlled-release systemsdiscussed in the review by Langer, 1990, Science 249:1527-1533) can beused.

When administered as a composition, a compound of the invention will beformulated with a suitable amount of a pharmaceutically acceptablevehicle or carrier so as to provide the form for proper administrationto the mammal. The term “pharmaceutically acceptable” means approved bya regulatory agency of the Federal or a state government or listed inthe U.S. Pharmacopeia or other generally recognized pharmacopeia for usein mammals, and more particularly in humans. The term “vehicle” refersto a diluent, adjuvant, excipient, or carrier with which a compound ofthe invention is formulated for administration to a mammal. Suchpharmaceutical vehicles can be liquids, such as water and oils,including those of petroleum, animal, vegetable or synthetic origin,such as peanut oil, soybean oil, mineral oil, sesame oil and the like.The pharmaceutical vehicles can be saline, gum acacia, gelatin, starchpaste, talc, keratin, colloidal silica, urea, and the like. In addition,auxiliary, stabilizing, thickening, lubricating and coloring agents maybe used. Preferably, when administered to a mammal, the compounds andcompositions of the invention and pharmaceutically acceptable vehicles,excipients, or diluents are sterile. An aqueous medium is a preferredvehicle when the compound of the invention is administeredintravenously, such as water, saline solutions, and aqueous dextrose andglycerol solutions.

The present compounds and compositions can take the form of capsules,tablets, pills, pellets, lozenges, powders, granules, syrups, elixirs,solutions, suspensions, emulsions, suppositories, or sustained-releaseformulations thereof, or any other form suitable for administration to amammal. In a preferred embodiment, the compounds and compositions of theinvention are formulated for administration in accordance with routineprocedures as a pharmaceutical composition adapted for oral orintravenous administration to humans. In one embodiment, thepharmaceutically acceptable vehicle is a hard gelatin capsule. Examplesof suitable pharmaceutical vehicles and methods for formulation thereofare described in Remington: The Science and Practice of Pharmacy,Alfonso R. Gennaro ed., Mack Publishing Co. Easton, Pa., 19th ed., 1995,Chapters 86, 87, 88, 91, and 92, incorporated herein by reference.

Compounds and compositions of the invention formulated for oraldelivery, are preferably in the form of capsules, tablets, pills, or anycompressed pharmaceutical form. Moreover, where in tablet or pill form,the compounds and compositions may be coated to delay disintegration andabsorption in the gastrointestinal tract thereby providing a sustainedaction over an extended period of time. Selectively permeable membranessurrounding an osmotically active driving compound are also suitable fororally administered compounds and compositions of the invention. Inthese later platforms, fluid from the environment surrounding thecapsule is imbibed by the driving compound that swells to displace theagent or agent composition through an aperture. These delivery platformscan provide an essentially zero order delivery profile as opposed to thespiked profiles of immediate release formulations. A time delay materialsuch as glycerol monostearate or glycerol stearate may also be used.Oral compositions can include standard vehicles, excipients, anddiluents, such as magnesium stearate, sodium saccharine, cellulose,magnesium carbonate, lactose, dextrose, sucrose, sorbitol, mannitol,starch, gum acacia, calcium silicate, microcrystalline cellulose,polyvinylpyrrolidinone, water, syrup, and methyl cellulose, theformulations can additionally include lubricating agents, such as talc,magnesium stearate, mineral oil, wetting agents, emulsifying andsuspending agents, preserving agents such as methyl- andpropylhydroxybenzoates. Such vehicles are preferably of pharmaceuticalgrade. Orally administered compounds and compositions of the inventioncan optionally include one or more sweetening agents, such as fructose,aspartame or saccharin; one or more flavoring agents such as peppermint,oil of wintergreen, or cherry; or one or more coloring agents to providea pharmaceutically palatable preparation.

A therapeutically effective dosage regimen for the treatment of aparticular disorder or condition will depend on its nature and severity,and can be determined by standard clinical techniques according to thejudgment of a medical practitioner. In addition, in vitro or in vivoassays can be used to help identify optimal dosages. Of course, theamount of a compound of the invention that constitutes a therapeuticallyeffective dose also depends on the administration route. In general,suitable dosage ranges for oral administration are about 0.001milligrams to about 20 milligrams of a compound of the invention perkilogram body weight per day, preferably, about 0.7 milligrams to about6 milligrams, more preferably, about 1.5 milligrams to about 4.5milligrams. In a preferred embodiment, a mammal, preferably, a human isorally administered about 0.01 mg to about 1000 mg of a compound of theinvention per day, more preferably, about 0.1 mg to about 300 mg perday, or about 1 mg to about 100 mg in single or divided doses. Thedosage amounts described herein refer to total amounts administered;that is, if more than one compound of the invention is administered, thepreferred dosages correspond to the total amount of the compounds of theinvention administered. Oral compositions preferably contain 10% to 95%of a compound of the invention by weight. Preferred unit oral-dosageforms include pills, tablets, and capsules, more preferably capsules.Typically such unit-dosage forms will contain about 0.01 mg, 0.1 mg, 1mg, 5 mg, 10 mg, 15 mg, 20 mg, 50 mg, 100 mg, 250 mg, or 500 mg of acompound of the invention, preferably, from about 5 mg to about 200 mgof compound per unit dosage.

In another embodiment, the compounds and compositions of the inventioncan be administered parenterally (e.g., by intramuscular, intrathecal,intravenous, and intraarterial routes), preferably, intravenously.Typically, compounds and compositions of the invention for intravenousadministration are solutions in sterile isotonic aqueous vehicles, suchas water, saline, Ringer's solution, or dextrose solution. Wherenecessary, the compositions may also include a solubilizing agent.Compositions for intravenous administration may optionally include alocal anesthetic such as lignocaine to ease pain at the site of theinjection. For intravenous administration, the compounds andcompositions of the invention can be supplied as a sterile, drylyophilized powder or water-free concentrate in a hermetically sealedcontainer, such as an ampule or sachette, the container indicating thequantity of active agent. Such a powder or concentrate is then dilutedwith an appropriate aqueous medium prior to intravenous administration.An ampule of sterile water, saline solution, or other appropriateaqueous medium can be provided with the powder or concentrate fordilution prior to administration. Or the compositions can be supplied inpre-mixed form, ready for administration. Where a compound orcomposition of the invention is to be administered by intravenousinfusion, it can be dispensed, for example, with an infusion bottlecontaining sterile pharmaceutical-grade water, saline, or other suitablemedium.

Rectal administration can be effected through the use of suppositoriesformulated from conventional carriers such as cocoa butter, modifiedvegetable oils, and other fatty bases. Suppositories can be formulatedby well-known methods using well-known formulations, for example seeRemington: The Science and Practice of Pharmacy, Alfonso R. Gennaro ed.,Mack Publishing Co. Easton, Pa., 19th ed., 1995, pp. 1591-1597,incorporated herein by reference

To formulate and administer topical dosage forms, well-known transdermaland intradermal delivery mediums such as lotions, creams, and ointmentsand transdermal delivery devices such as patches can be used (Ghosh, T.K.; Pfister, W. R.; Yum, S. I. Transdermal and Topical Drug DeliverySystems, Interpharm Press, Inc. p. 249-297, incorporated herein byreference). For example, a reservoir type patch design can comprise abacking film coated with an adhesive, and a reservoir compartmentcomprising a compound or composition of the invention, that is separatedfrom the skin by a semipermeable membrane (e.g., U.S. Pat. No.4,615,699, incorporated herein by reference). The adhesive coatedbacking layer extends around the reservoir's boundaries to provide aconcentric seal with the skin and hold the reservoir adjacent to theskin.

Mucosal dosage forms of the invention include, but are not limited to,ophthalmic solutions, sprays and aerosols, or other forms known to oneof skill in the art. See, e.g., Remington's Pharmaceutical Sciences,18th eds., Mack Publishing, Easton Pa. (1990); and Introduction toPharmaceutical Dosage Forms, 4th ed., Lea & Febiger, Philadelphia(1985). Dosage forms suitable for treating mucosal tissues within theoral cavity can be formulated as mouthwashes or as oral gels. In oneembodiment, the aerosol comprises a carrier. In another embodiment, theaerosol is carrier free.

The compounds of the invention may also be administered directly to thelung by inhalation. For administration by inhalation, a compound of theinvention can be conveniently delivered to the lung by a number ofdifferent devices. For example, a Metered Dose Inhaler (“MDI”) whichutilizes canisters that contain a suitable low boiling propellant, e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas can beused to deliver a compound of formula I directly to the lung. MDIdevices are available from a number of suppliers such as 3M Corporation,Aventis, Boehringer Ingleheim, Forest Laboratories, Glaxo-Wellcome,Schering Plough and Vectura.

Alternatively, a Dry Powder Inhaler (DPI) device can be used toadminister a compound of the invention to the lung (See, e.g., Raleighet al., Proc. Amer. Assoc. Cancer Research Annual Meeting, 1999, 40,397, which is herein incorporated by reference). DPI devices typicallyuse a mechanism such as a burst of gas to create a cloud of dry powderinside a container, which can then be inhaled by the patient. DPIdevices are also well known in the art and can be purchased from anumber of vendors which include, for example, Fisons, Glaxo-Wellcome,Inhale Therapeutic Systems, ML Laboratories, Qdose and Vectura. Apopular variation is the multiple dose DPI (“MDDPI”) system, whichallows for the delivery of more than one therapeutic dose. MDDPI devicesare available from companies such as AstrZeneca, GlaxoWellcome, IVAX,Schering Plough, SkyePharma and Vectura. For example, capsules andcartridges of gelatin for use in an inhaler or insufflator can beformulated containing a powder mix of the compound and a suitable powderbase such as lactose or starch for these systems.

Another type of device that can be used to deliver a compound of theinvention to the lung is a liquid spray device supplied, for example, byAradigm Corporation. Liquid spray systems use extremely small nozzleholes to aerosolize liquid drug formulations that can then be directlyinhaled into the lung.

In a preferred embodiment, a nebulizer device is used to deliver acompound of the invention to the lung. Nebulizers create aerosols fromliquid drug formulations by using, for example, ultrasonic energy toform fine particles that can be readily inhaled (See e.g., Verschoyle etal., British J Cancer, 1999, 80, Suppl 2, 96, which is hereinincorporated by reference). Examples of nebulizers include devicessupplied by Sheffield/Systemic Pulmonary Delivery Ltd. (See, Armer etal., U.S. Pat. No. 5,954,047; van der Linden et al., U.S. Pat. No.5,950,619; van der Linden et al., U.S. Pat. No. 5,970,974, which areherein incorporated by reference), Aventis and Batelle PulmonaryTherapeutics

In a particularly preferred embodiment, an electrohydrodynamic (“EHD”)aerosol device is used to deliver a compound of the invention to thelung. EHD aerosol devices use electrical energy to aerosolize liquiddrug solutions or suspensions (see e.g., Noakes et al., U.S. Pat. No.4,765,539; Coffee, U.S. Pat. No., 4,962,885; Coffee, PCT Application, WO94/12285; Coffee, PCT Application, WO 94/14543; Coffee, PCT Application,WO 95/26234, Coffee, PCT Application, WO 95/26235, Coffee, PCTApplication, WO 95/32807, which are herein incorporated by reference).The electrochemical properties of the compound of formula I formulationmay be important parameters to optimize when delivering this drug to thelung with an EHD aerosol device and such optimization is routinelyperformed by one of skill in the art. EHD aerosol devices may moreefficiently delivery drugs to the lung than existing pulmonary deliverytechnologies.

Liquid drug formulations suitable for use with nebulizers and liquidspray devices and EHD aerosol devices will typically include afluoroalkoxy-substituted 1,3-dihydro-isoindolyl compound with apharmaceutically acceptable carrier. Preferably, the pharmaceuticallyacceptable carrier is a liquid such as alcohol, water, polyethyleneglycol or a perfluorocarbon. Optionally, another material may be addedto alter the aerosol properties of the solution or suspension of acompound of the invention. Preferably, this material is liquid such asan alcohol, glycol, polyglycol or a fatty acid. Other methods offormulating liquid drug solutions or suspension suitable for use inaerosol devices are known to those of skill in the art (See, e.g.,Biesalski, U.S. Pat. No. 5,112,598; Biesalski, U.S. Pat. No. 5,556,611,which are herein incorporated by reference) A compound of formula I canalso be formulated in rectal or vaginal compositions such assuppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, a compound of theinvention can also be formulated as a depot preparation. Such longacting formulations can be administered by implantation (for examplesubcutaneously or intramuscularly) or by intramuscular injection. Thus,for example, the compounds can be formulated with suitable polymeric orhydrophobic materials (for example, as an emulsion in an acceptable oil)or ion exchange resins, or as sparingly soluble derivatives, forexample, as a sparingly soluble salt.

Alternatively, other pharmaceutical delivery systems can be employed.Liposomes and emulsions are well known examples of delivery vehiclesthat can be used to deliver compounds of the inventions. Certain organicsolvents such as dimethylsulfoxide can also be employed, althoughusually at the cost of greater toxicity. A compound of the invention canalso be delivered in a controlled release system. In one embodiment, apump can be used (Sefton, CRC Crit. Ref Biomed Eng., 1987, 14, 201;Buchwald et al., Surgery, 1980, 88, 507; Saudek et al., N. Engl. Med,1989, 321, 574). In another embodiment, polymeric materials can be used(see Medical Applications of Controlled Release, Langer and Wise (eds.),CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability,Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, NewYork (1984); Ranger and Peppas, J Macromol. Sci. Rev. Macromol. Chem.,1983, 23, 61; see also Levy et al., Science 1985, 228, 190; During etal., Ann. Neurol., 1989,25,351; Howard et al., 1989, J. Neurosurg. 71,105). In yet another embodiment, a controlled-release system can beplaced in proximity of the target of the compounds of the invention,e.g., the lung, thus requiring only a fraction of the systemic dose(see, e.g., Goodson, in Medical Applications of Controlled Release,supra, vol. 2, pp. 115 (1984)). Other controlled-release system can beused (see e.g. Langer, Science, 1990, 249, 1527).

Suitable excipients (e.g., carriers and diluents) and other materialsthat can be used to provide mucosal dosage foul's encompassed by thisinvention are well known to those skilled in the pharmaceutical arts,and depend on the particular site or method which a given pharmaceuticalcomposition or dosage form will be administered. With that fact in mind,typical excipients include, but are not limited to, water, ethanol,ethylene glycol, propylene glycol, butane-1,3-diol, isopropyl myristate,isopropyl palmitate, mineral oil, and mixtures thereof, which arenon-toxic and pharmaceutically acceptable. Examples of such additionalingredients are well known in the art. See, e.g., Remington'sPharmaceutical Sciences, 18th eds., Mack Publishing, Easton Pa. (1990).

The pH of a pharmaceutical composition or dosage form, or of the tissueto which the pharmaceutical composition or dosage form is applied, canalso be adjusted to improve delivery of one or more active ingredients.Similarly, the polarity of a solvent carrier, its ionic strength, ortonicity can be adjusted to improve delivery. Compounds such asstearates can also be added to pharmaceutical compositions or dosageforms to advantageously alter the hydrophilicity or lipophilicity of oneor more active ingredients so as to improve delivery. In this regard,stearates can serve as a lipid vehicle for the formulation, as anemulsifying agent or surfactant, and as a delivery-enhancing orpenetration-enhancing agent. Different salts, hydrates or solvates ofthe active ingredients can be used to further adjust the properties ofthe resulting composition.

The invention also provides pharmaceutical packs or kits comprising oneor more containers filled with one or more compounds of the invention.Optionally associated with such container(s) can be a notice in the formprescribed by a governmental agency regulating the manufacture, use orsale of pharmaceuticals or biological products, which notice reflectsapproval by the agency of manufacture, use or sale for humanadministration. In one embodiment, the kit contains more than onecompound of the invention. In another embodiment, the kit comprises acompound of the invention and another biologically active agent.

The compounds of the invention are preferably assayed in vitro and invivo, for the desired therapeutic or prophylactic activity, prior to usein humans. For example, in vitro assays can be used to determine whetheradministration of a specific compound of the invention or a combinationof compounds of the invention is preferred. The compounds andcompositions of the invention may also be demonstrated to be effectiveand safe using animal model systems. Other methods will be known to theskilled artisan and are within the scope of the invention.

4.5 Combination Therapy

In certain embodiments, a compound of the invention is administered to amammal, preferably, a human concurrently with one or more othertherapeutic agents, or with one or more other compounds of theinvention, or with both. By “concurrently” it is meant that a compoundof the invention and the other agent are administered to a mammal in asequence and within a time interval such that the compound of theinvention can act together with the other agent to provide an increasedor synergistic benefit than if they were administered otherwise. Forexample, each component may be administered at the same time orsequentially in any order at different points in time; however, if notadministered at the same time, they should be administered sufficientlyclosely in time so as to provide the desired treatment effect.Preferably, all components are administered at the same time, and if notadministered at the same time, preferably, they are all administeredfrom about 6 hours to about 12 hours apart from one another.

When used in combination with other therapeutic agents, the compounds ofthe invention and the therapeutic agent can act additively or, morepreferably, synergistically. In one embodiment, a compound or acomposition of the invention is administered concurrently with anothertherapeutic agent in the same pharmaceutical composition. In anotherembodiment, a compound or a composition of the invention is administeredconcurrently with another therapeutic agent in separate pharmaceuticalcompositions. In still another embodiment, a compound or a compositionof the invention is administered prior or subsequent to administrationof another therapeutic agent. As many of the disorders for which thecompounds and compositions of the invention are useful in treating arechronic disorders, in one embodiment combination therapy involvesalternating between administering a compound or a composition of theinvention and a pharmaceutical composition comprising anothertherapeutic agent, e.g., to minimize the toxicity associated with aparticular drug. In certain embodiments, when a composition of theinvention is administered concurrently with another therapeutic agentthat potentially produces adverse side effects including, but notlimited to toxicity, the therapeutic agent can advantageously beadministered at a dose that falls below the threshold that the adverseside effect is elicited. Additional therapeutic agents include, but arenot limited to, hematopoietic growth factors, cytokines, anti-canceragents, antibiotics, immunosuppressive agents, steroids, antihistamines,lukatriene inhibitors and other therapeutics discussed herein.

Preferred additional therapeutic agents include, but are not limited to,Remicade™, docetaxel, Celecoxib™, melphalan, dexamethasone, steroids,gemcitabine, cisplatinum, temozolomide, etoposide, cyclophosphamide,temodar, carboplatin, procarbazine, gliadel, tamoxifen, topotecan,methotrexate, Arisa®, Taxol™, taxotere, fluorouracil, leucovorin,irinotecan, xeloda, CPT-11, interferon alpha, pegylated interferonalpha, capecitabine, cisplatin, thiotepa, fludarabine, carboplatin,liposomal daunorubicin, cytarabine, doxetaxol, pacilitaxel, vinblastine,IL-2, GM-CSF, dacarbazine, vinorelbine, zoledronic acid, palmitronate,biaxin, busulphan, prednisone, bisphosphonate, arsenic trioxide, PEGINTRON-A, doxil, vincristine, decadron, doxorubicin, paclitaxel,ganciclovir, adriamycin, estramustine, Emcyt, sulindac, and etoposide.

The invention further encompasses mutants and derivatives (e.g.,modified forms) of naturally occurring proteins that exhibit, in vivo,at least some of the pharmacological activity of the proteins upon whichthey are based. Examples of mutants include, but are not limited to,proteins that have one or more amino acid residues that differ from thecorresponding residues in the naturally occurring forms of the proteins.Also encompassed by the term “mutants” are proteins that lackcarbohydrate moieties normally present in their naturally occurringforms (e.g., nonglycosylated forms). Examples of derivatives include,but are not limited to, pegylated derivatives and fusion proteins, suchas proteins formed by fusing IgG1 or IgG3 to the protein or activeportion of the protein of interest. See, e.g., Penichet, M. L. andMorrison, S. L., J. Immunol. Methods 248:91-101 (2001).

Recombinant and mutated forms of G-CSF can be prepared as described inU.S. Pat. Nos. 4,810,643; 4,999,291; 5,528,823; and 5,580,755; all ofwhich are incorporated herein by reference. Recombinant and mutatedforms of GM-CSF can be prepared as described in U.S. Pat. Nos.5,391,485; 5,393,870; and 5,229,496; all of which are incorporatedherein by reference. In fact, recombinant forms of G-CSF and GM-CSF arecurrently sold in the United States for the treatment of symptomsassociated with specific chemotherapies. A recombinant form of G-CSFknown as filgrastim is sold in the United States under the trade nameNEUPOGEN®, and is indicated to decrease the incidence of infection, asmanifested by febrile neutropenia, in patients with nonmyeloidmalignancies receiving myelosuppressive anti-cancer drugs associatedwith a significant incidence of severe neutropenia with fever.Physicians' Desk Reference, 587-592 (56^(th) ed., 2002). A recombinantform of GM-CSF known as sargramostim is also sold in the United Statesunder the trade name LEUKINE®. LEUKINE® is indicated for use followinginduction chemotherapy in older adult patients with acute myelogenousleukemia (AML) to shorten time to neutrophil recovery. Physicians' DeskReference, 1755-1760 (56^(th) ed., 2002). A recombinant form of EPOknown as epoetin alfa is sold in the United States under the trade nameEPOGEN®. EPOGEN® is used to stimulate red cell production by stimulatingdivision and maturation of committed red cell precursor cells.Physicians' Desk Reference, 582-587 (56^(th) ed., 2002).

A growth-factor or cytokine such as G-CSF, GM-CSF and EPO can also beadministered in the form of a vaccine. For example, vaccines thatsecrete, or cause the secretion of, cytokines such as G-CSF and GM-CSFcan be used in the methods, pharmaceutical compositions, and kits of theinvention. See, e.g., Emens, L. A., et al., Curr. Opinion Mol. Ther.3(1):77-84 (2001).

Examples of anti-cancer drugs that can be used in the variousembodiments of the invention, including the methods, dosing regimens,cocktails, pharmaceutical compositions and dosage forms and kits of theinvention, include, but are not limited to: acivicin; aclarubicin;acodazole hydrochloride; acronine; adozelesin; aldesleukin; altretamine;ambomycin; ametantrone acetate; amsacrine; anastrozole; anthramycin;asparaginase; asperlin; azacitidine; azetepa; azotomycin; batimastat;benzodepa; bicalutamide; bisantrene hydrochloride; bisnafide dimesylate;bizelesin; bleomycin sulfate; brequinar sodium; bropirimine; busulfan;cactinomycin; calusterone; caracemide; carbetimer; carboplatin;carmustine; carubicin hydrochloride; carzelesin; cedefingol; celecoxib;chlorambucil; cirolemycin; cisplatin; cladribine; crisnatol mesylate;cyclophosphamide; cytarabine; dacarbazine; dactinomycin; daunorubicinhydrochloride; decitabine; dexormaplatin; dezaguanine; dezaguaninemesylate; diaziquone; dacarbazine; docetaxel; doxorubicin; doxorubicinhydrochloride; droloxifene; droloxifene citrate; dromostanolonepropionate; duazomycin; edatrexate; eflornithine hydrochloride;elsamitrucin; enloplatin; enpromate; epipropidine; epirubicinhydrochloride; erbulozole; esorubicin hydrochloride; estramustine;estramustine phosphate sodium; etanidazole; etoposide; etoposidephosphate; etoprine; fadrozole hydrochloride; fazarabine; fenretinide;floxuridine; fludarabine phosphate; fluorouracil; fluorocitabine;fosquidone; fostriecin sodium; gemcitabine; gemcitabine hydrochloride;hydroxyurea; idarubicin hydrochloride; ifosfamide; ilmofosine;interleukin II (including recombinant interleukin II, or rIL2),interferon alfa-2a; interferon alfa-2b; interferon alfa-n1; interferonalfa-n3; interferon beta-I a; interferon gamma-I b; iproplatin;irinotecan; irinotecan hydrochloride; lanreotide acetate; letrozole;leuprolide acetate; liarozole hydrochloride; lometrexol sodium;lomustine; losoxantrone hydrochloride; masoprocol; maytansine;mechlorethamine hydrochloride; megestrol acetate; melengestrol acetate;melphalan; menogaril; mercaptopurine; methotrexate; methotrexate sodium;metoprine; meturedepa; mitindomide; mitocarcin; mitocromin; mitogillin;mitomalcin; mitomycin; mitosper; mitotane; mitoxantrone hydrochloride;mycophenolic acid; nocodazole; nogalamycin; ormaplatin; oxisuran;paclitaxel; pegaspargase; peliomycin; pentamustine; peplomycin sulfate;perfosfamide; pipobroman; piposulfan; piroxantrone hydrochloride;plicamycin; plomestane; porfimer sodium; porfiromycin; prednimustine;procarbazine hydrochloride; puromycin; puromycin hydrochloride;pyrazofurin; riboprine; safingol; safingol hydrochloride; semustine;simtrazene; sparfosate sodium; sparsomycin; spirogermaniumhydrochloride; spiromustine; spiroplatin; streptonigrin; streptozocin;sulofenur; talisomycin; tecogalan sodium; taxotere; tegafur;teloxantrone hydrochloride; temoporfin; teniposide; teroxirone;testolactone; thiamiprine; thioguanine; thiotepa; tiazofurin;tirapazamine; toremifene citrate; trestolone acetate; triciribinephosphate; trimetrexate; trimetrexate glucuronate; triptorelin;tubulozole hydrochloride; uracil mustard; uredepa; vapreotide;verteporfin; vinblastine sulfate; vincristine sulfate; vindesine;vindesine sulfate; vinepidine sulfate; vinglycinate sulfate;vinleurosine sulfate; vinorelbine tartrate; vinrosidine sulfate;vinzolidine sulfate; vorozole; zeniplatin; zinostatin; and zorubicinhydrochloride. Other anti-cancer drugs include, but are not limited to:20-epi-1,25 dihydroxyvitamin D3; 5-ethynyluracil; abiraterone;aclarubicin; acylfulvene; adecypenol; adozelesin; aldesleukin; ALL-TKantagonists; altretamine; ambamustine; amidox; amifostine;aminolevulinic acid; amrubicin; amsacrine; anagrelide; anastrozole;andrographolide; angiogenesis inhibitors; antagonist D; antagonist G;antarelix; anti-dorsalizing morphogenetic protein-1; antiandrogen,prostatic carcinoma; antiestrogen; antineoplaston; antisenseoligonucleotides; aphidicolin glycinate; apoptosis gene modulators;apoptosis regulators; apurinic acid; ara-CDP-DL-PTBA; argininedeaminase; asulacrine; atamestane; atrimustine; axinastatin 1;axinastatin 2; axinastatin 3; azasetron; azatoxin; azatyrosine; baccatinIII derivatives; balanol; batimastat; BCR/ABL antagonists;benzochlorins; benzoylstaurosporine; beta lactam derivatives;beta-alethine; betaclamycin B; betulinic acid; bFGF inhibitor;bicalutamide; bisantrene; bisaziridinylspermine; bisnafide; bistrateneA; bizelesin; breflate; bropirimine; budotitane; buthionine sulfoximine;calcipotriol; calphostin C; camptothecin derivatives; canarypox IL-2;capecitabine; carboxamide-amino-triazole; carboxyamidotriazole;cartilage derived inhibitor; carzelesin; casein kinase inhibitors(ICOS); castanospermine; cecropin B; cetrorelix; chlorins;chloroquinoxaline sulfonamide; cicaprost; cis-porphyrin; cladribine;clomifene analogues; clotrimazole; collismycin A; collismycin B;combretastatin A4; combretastatin analogue; conagenin; crambescidin 816;crisnatol; cryptophycin 8; cryptophycin A derivatives; curacin A;cyclopentanthraquinones; cycloplatam; cypemycin; cytarabine ocfosfate;cytolytic factor; cytostatin; dacliximab; decitabine; dehydrodidemnin B;deslorelin; dexamethasone; dexifosfamide; dexrazoxane; dexverapamil;diaziquone; didemnin B; diethylnorspermine; dihydro-5-azacytidine;dihydrotaxol; dioxamycin; diphenyl spiromustine; docetaxel; docosanol;dolasetron; doxifluridine; doxorubicin; droloxifene; dronabinol;duocarmycin SA; ebselen; ecomustine; edelfosine; edrecolomab;eflornithine; elemene; emitefur; epirubicin; epristeride; estramustineanalogue; estrogen agonists; estrogen antagonists; etanidazole;etoposide phosphate; exemestane: fadrozole; fazarabine; fenretinide;filgrastim: finasteride; flavopiridol; flezelastine; fluasterone;fludarabine; fluorodaunorunicin hydrochloride; forfenimex; formestane;fostriecin; fotemustine; gadolinium texaphyrin; gallium nitrate;galocitabine; ganirelix; gelatinase inhibitors; gemcitabine; glutathioneinhibitors; hepsulfam; heregulin; hexamethylene bisacetamide; hypericin;ibandronic acid; idarubicin; idoxifene; idramantone; ilmofosine;ilomastat; imiquimod; immunostimulant peptides; insulin-like growthfactor-1 receptor inhibitor; interferon agonists; interferons;interleukins; iobenguane; iododoxorubicin; ipomeanol, 4-; iroplact;irsogladine; isobengazole; isohomohalicondrin B; itasetron;jasplakinolide; kahalalide F; lamellarin-N triacetate; lanreotide;leinamycin; lenograstim; lentinan sulfate; leptolstatin; letrozole;leukemia inhibiting factor; leukocyte alpha interferon;leuprolide+estrogen+progesterone; leuprorelin; levamisole; liarozole;linear polyamine analogue; lipophilic disaccharide peptide; lipophilicplatinum compounds; lissoclinamide 7; lobaplatin; lombricine;lometrexol; lonidamine; losoxantrone; loxoribine; lurtotecan; lutetiumtexaphyrin; lysofylline; lytic peptides; maitansine; mannostatin A;marimastat; masoprocol; maspin; matrilysin inhibitors; matrixmetalloproteinase inhibitors; menogaril; merbarone; meterelin;methioninase; metoclopramide; MIF inhibitor; mifepristone; miltefosine;mirimostim; mitoguazone; mitolactol; mitomycin analogues; mitonafide;mitotoxin fibroblast growth factor-saporin; mitoxantrone; mofarotene;molgramostim; Erbitux, human chorionic gonadotrophin; mopidamol; mustardanticancer agent; mycaperoxide B; mycobacterial cell wall extract;myriaporone; N-acetyldinaline; N-substituted benzamides; nafarelin;nagrestip; naloxone+pentazocine; napavin; naphterpin; nartograstim;nedaplatin; nemorubicin; neridronic acid; nilutamide; nisamycin; nitricoxide modulators; nitroxide antioxidant; nitrullyn; O⁶-benzylguanine;octreotide; okicenone; oligonucleotides; onapristone; ondansetron;ondansetron; oracin; oral cytokine inducer; ormaplatin; osaterone;oxaliplatin; oxaunomycin; paclitaxel; paclitaxel analogues; paclitaxelderivatives; palauamine; palmitoylrhizoxin; pamidronic acid;panaxytriol; panomifene; parabactin; pazelliptine; pegaspargase;peldesine; pentosan polysulfate sodium; pentostatin; pentrozole;perflubron; perfosfamide; perillyl alcohol; phenazinomycin;phenylacetate; phosphatase inhibitors; picibanil; pilocarpinehydrochloride; pirarubicin; piritrexim; placetin A; placetin B;plasminogen activator inhibitor; platinum complex; platinum compounds;platinum-triamine complex; porfimer sodium; porfiromycin; prednisone;propyl bis-acridone; prostaglandin J2; proteasome inhibitors; proteinA-based immune modulator; protein kinase C inhibitor; protein kinase Cinhibitors, microalgal; protein tyrosine phosphatase inhibitors; purinenucleoside phosphorylase inhibitors; purpurins; pyrazoloacridine;pyridoxylated hemoglobin polyoxyethylene conjugate; raf antagonists;raltitrexed; ramosetron; ras farnesyl protein transferase inhibitors;ras inhibitors; ras-GAP inhibitor; retelliptine demethylated; rhenium Re186 etidronate; rhizoxin; ribozymes; RII retinamide; rohitukine;romurtide; roquinimex; rubiginone B1; ruboxyl; safingol; saintopin;SarCNU; sarcophytol A; sargramostim; Sdi 1 mimetics; semustine;senescence derived inhibitor 1; sense oligonucleotides; signaltransduction inhibitors; sizofuran; sobuzoxane; sodium borocaptate;sodium phenylacetate; solverol; somatomedin binding protein; sonermin;sparfosic acid; spicamycin D; spiromustine; splenopentin; spongistatin1; squalamine; stipiamide; stromelysin inhibitors; sulfinosine;superactive vasoactive intestinal peptide antagonist; suradista;suramin; swainsonine; tallimustine; tamoxifen methiodide; tauromustine;tazarotene; tecogalan sodium; tegafur; tellurapyrylium; telomeraseinhibitors; temoporfin; teniposide; tetrachlorodecaoxide; tetrazomine;thaliblastine; thiocoraline; thrombopoietin; thrombopoietin mimetic;thymalfasin; thymopoietin receptor agonist; thymotrinan; thyroidstimulating hormone; tin ethyl etiopurpurin; tirapazamine; titanocenebichloride; topsentin; toremifene; translation inhibitors; tretinoin;triacetyluridine; triciribine; trimetrexate; triptorelin; tropisetron;turosteride; tyrosine kinase inhibitors; tyrphostins; UBC inhibitors;ubenimex; urogenital sinus-derived growth inhibitory factor; urokinasereceptor antagonists; vapreotide; variolin B; velaresol; veramine;verdins; verteporfin; vinorelbine; vinxaltine; vitaxin; vorozole;zanoterone; zeniplatin; zilascorb; and zinostatin stimalamer.

In one embodiment of the invention, the compounds of the invention canbe used, not only to directly treat the disorder, but also to reduce thedose or toxicity of another chemotherapeutic. For example, the compoundsof the invention can be administered to reduce gastrointestinal toxicityassociated with a topoisomerase inhibitor, such as irinotecan.

4.6 Biological Assays

Compounds having PDE 4, TNF-α, and MMP inhibitory activity can beassayed using methods commonly known in the art including, but notlimited to, enzyme immunoassay, radioimmunoassay, immunoelectrophoresis,and affinity labeling. Further assays which can be utilized includeLPS-induced TNF and PDE4 enzymatic assays and the methods set out inInternational Patent Publication Nos. WO 01/90076 A1 WO 01/34606 A1 eachof which are incorporated herein in their entireties by reference.

PBMC from normal donors are obtained by Ficoll-Hypaque densitycentrifugation. Cells are cultured in RPMI supplemented with 10%, AB+serum, 2 mM L-glutamine, 100 U/mL penicillin and 100 mg/mL streptomycin.

The test compounds are dissolved in dimethylsulfoxide (Sigma Chemical),further dilutions are done in supplemented RPMI. The finaldimethylsulfoxide concentration in the presence or absence of drug inthe PBMC suspensions is 0.25 wt %. The test compounds are assayed athalf-log dilutions starting in 50 mg/mL. The test compounds are added toPBMC (106 cells/mL) in 96 wells plates one hour before the addition ofLPS.

PBMC (106 cells/mL) in the presence or absence of test compounds arestimulated by treatment with 1 mg/mL of LPS from Salmonella minnesotaR595 (List Biological Labs, Campbell, Calif.). Cells are then incubatedat 37 □C for 18-20 hours. Supernatants are harvested and assayedimmediately for TNFα levels or kept frozen at −70 □C (for not more than4 days) until assayed.

The concentration of TNFα in the supernatant is determined by human TNFαELISA kits (ENDOGEN, Boston, Mass.) according to the manufacturer'sdirections.

Phosphodiesterase can be determined in conventional models. For example,using the method of Hill and Mitchell, U937 cells of the humanpromonocytic cell line are grown to 1×106 cells/mL and collected bycentrifugation. A cell pellet of 1×109 cells is washed in phosphatebuffered saline and then frozen at −70□C for later purification orimmediately lysed in cold homogenization buffer (20 mM Tris-HCl, pH 7.1,3 mM 2-mercaptoethanol, 1 mM magnesium chloride, 0.1 mM ethyleneglycol-bis-(β-aminoethyl ether)-N,N,N═,N=-tetraacetic acid (EGTA), 1 μMphenyl-methylsulfonyl fluoride (PMSF), and 1 μg/mL leupeptin). Cells arehomogenized with 20 strokes in a Dounce homogenizer and supernatantcontaining the cytosolic fraction are obtained by centrifugation. Thesupernatant then is loaded onto a Sephacryl S-200 column equilibrated inhomogenization buffer. Phosphodiesterase is eluted in homogenizationbuffer at a rate of approximately 0.5 mL/min and fractions are assayedfor phosphodiesterase activity −/+ rolipram. Fractions containingphosphodiester-ase activity (rolipram sensitive) are pooled andaliquoted for later use.

The phosphodiesterase assay is carried out in a total volume of 100 μlcontaining various concentration of test compounds, 50 mM Tris-HCl, pH7.5, 5 mM magnesium chloride, and 1 μM cAMP of which 1% was 3H cAMP.Reactions are incubated at 30□C. for 30 minutes and terminated byboiling for 2 minutes. The amount of phosphodiesterase IV containingextract used for these experiments is predetermined such that reactionsare within the linear range and consumed less than 15% of the totalsubstrate. Following termination of reaction, samples are chilled at 4□C and then treated with 10 μl 10 mg/mL snake venom for 15 min at 30 □C.Unused substrate then is removed by adding 200 μl of a quaternaryammonium ion exchange resin (AG1-X8, BioRad) for 15 minutes. Samplesthen are spun at 3000 rpm, 5 min and 50 μl of the aqueous phase aretaken for counting. Each data point is carried out in duplicate andactivity is expressed as percentage of control. The IC50 of the compoundthen is determined from dose response curves of a minimum of threeindependent experiments.

The following examples are offered by way of illustration and are notintended to limit the scope of the invention.

5. EXAMPLES

Reagents and solvents used below can be obtained from commercial sourcessuch as Aldrich Chemical Co. (Milwaukee, Wis., USA). ¹H-NMR and ¹³C-NMRspectra were recorded on a Bruker AC 250 MHz NMR spectrometer.Significant peaks are tabulated in the order: chemical shift,multiplicity (s, singlet; d, doublet; t, triplet; q, quartet; m,multiplet; br s, broad singlet), coupling constant(s) in Hertz (Hz) andnumber of protons.

5.1 Example 1 4-Difluoromethoxy-3-hydroxy-benzaldehyde

A vigorously stirred mixture of 3,4-dihydroxybenzaldehyde (25 g, 0.18mol) and potassium carbonate (25 g 0.18 mol) in dimethylformamide (125ml) was heated under an atmosphere of chlorodifluoromethane using a —78°C. condenser at 100° C. for 5.5 hours. The mixture was allowed to cool,was acidified to pH 5-6 with concentrated hydrochloric acid and wasconcentrated under reduced pressure. The residue was partitioned betweenether and 3N aqueous hydrochloric acid and extracted five times withether. The organic extract was dried over magnesium sulfate and thesolvent was removed in vacuo. The residue was purified by flashchromatography, eluting with 2:1 hexane/ethyl acetate, to provide ayellow solid, which was triturated with ethyl acetate/hexane to give4-difluoromethoxy-3-hydroxy-benzaldehyde as a white solid (5 g, 15%).¹HNMR (DMSO-d₆): δ 7.22 (t, J_(H-F)=75 Hz, 1H), 7.31 (d, J=10 Hz, 1H),7.41-7.43 (m, 2H), 9.87 (s, 1H), 10.50 (s, 1H).

5.2 Example 2 3-Cyclopropylmethoxy-4-difluoromethoxy -benzaldehyde

To a mixture of 4-difluoromethoxy-3-hydroxy-benzaldehyde (5.0 g, 27mmol) and potassium carbonate (5.5 g, 40 mmol) in dimethylformamide (30ml) under inert atmosphere at 60° C. was added bromoethylcyclopropane (5g, 37 mmol). The mixture was stirred and heated at 65° C. After 1.5hour, the mixture was allowed to cool and was filtered. The filtrate wasconcentrated under reduced pressure. The mixture was extracted withethyl acetate (2×25 ml) and water (25 ml). The organic layer was washedwith water (25 ml), brine (25 ml) and dried over magnesium sulfate. Thesolvent was removed in vacuo to give3-cyclopropylmethoxy-4-difluoromethoxy-benzaldehyde as an oil (6.4 g,100%). ¹HNMR (CDCl₃):

0.38-0.44 (m, 2H), 0.62-0.75 (m, 2H), 1.15-1.36 (m, 1H), 3.98 (d, J=4.5Hz, 2H), 6.78 (t, J_(H-F)=75 Hz, 1H), 7.30-7.50 (m. 3H), 9.96 (s, 1H).

5.3 Example 3 3-amino-3-(3-cyclopropylmethoxy-4-difluoromethoxy-phenyl)-propionic acid

To a mixture of 3-cyclopropylmethoxy-4-difluoromethoxy-benzaldehyde (6.4g, 27 mmol) and ammonium acetate (4.3 g, 55 mmol) in 95% ethanol (30 ml)under nitrogen atmosphere at 40° C. was added malonic acid (2.9 g, 28mmol). The mixture was stirred and heated at reflux temperature for 20hours. The mixture was allowed to cool and was filtered to give3-cyclopropylmethoxy-4-difluoromethoxy-benzaldehyde as a white solid(4.3 g, 52%). ¹HNMR (DMSO-d₆): δ 0.31-0.35 (m, 2H), 0.52-0.58 (m, 2H),1.15-1.36 (m, 1H), 2.33-2.37 (m, 2H), 3.87 (d, J=7.5 Hz, 2H), 4.20-4.26(m, 1H), 6.94-6.98 (m, 1H), 7.03 (t, J_(H-F)=75 Hz, 1H), 7.13 (d, J=10Hz, 1H), 7.23 (d, J=2.5 Hz, 1H).

5.4 Example 4 3-(4-Acetylamino-1,3-dioxo-1,3-dihydro-isoindol-2-yl)-3-(3-cyclopropylmethoxy-4-difluoromethoxy-phenyl)-propionic acid

To a solution of3-amino-3-(3-cyclopropylmethoxy-4-difluoromethoxy-phenyl)-propionic acid(500 mg, 1.0 mmol) in acetic acid (10 ml) was added 3-acetamido-phthalicanhydride (390 mg, 1.9 mmol) and sodium acetate (160 mg, 1.9 mmol). Themixture was heated at reflux temperature overnight. The solvent wasremoved in vacuo. The resulted oil was extracted with ethyl acetate (50ml) and water (30 ml). The organic layer was washed with water (30ml×4), brine (30 ml) and dried over magnesium sulfate. The solvent wasremoved in vacuo and the resulted oil was slurried with ether for 2hours. The suspension was filtered to give3-(4-acetylamino-1,3-dioxo-1,3-dihydro-isoindol-2-yl)-3-(3-cyclopropylmethoxy-4-difluoromethoxy-phenyl)-propionicacid as a white solid (720 mg, 85%). ¹HNMR (CDCl₃):

0.35-0.39 (m, 2H), 0.64-0.68 (m, 2H), 1.15-1.36 (m, 1H), 2.27 (s, 3H),3.24 (dd, J=5.8, 17 Hz, 1H), 3.82 (dd, J=10, 17 Hz, 1H), 3.88 (d, J=8.3Hz, 2H), 5.70 (dd, J=5.8, 10 Hz, 1H), 6.59 (t, J_(H-F)=75 Hz, 1H),7.06-7.14 (m, 3H), 7.48 (d, J=7.3 Hz, 1H), 7.65 (t, J=7.5 Hz, 1H), 8.76(d, J=8.3 Hz, 1H), 9.47 (s, 1H).

5.5 Example 53-(4-Acetylamino-1,3-dioxo-1,3-dihydro-isoindol-2-yl)-3-(3-cyclopropylmethoxy-4-difluoromethoxy-phenyl)-N,N-dimethyl-propionamide

To a solution of3-(4-acetylamino-1,3-dioxo-1,3-dihydro-isoindol-2-yl)-3-(3-cyclopropylmethoxy-4-difluoromethoxy-phenyl)-propionicacid (470 mg, 1.04 mmol) in tetrahydrofurane (10 ml) was addedcarbonyldiimidazole (250 mg, 1.56 mmol) at room temperature. Thesolution was stirred at room temperature for 2 hours. To the mixture wasadded dimethylamine (2.0N in THF, 1.0 ml, 2.0 mmol). The resultedmixture was stirred at room temperature for 3 hours. Water (20 ml) wasadded to the reaction mixture. The solvent was removed in vacuo. Theresulted mixture was dissolved in ethyl acetate (30 ml) and extractedwith water (20 ml). The organic layer was washed with saturated sodiumbicarbonate solution (3×20 ml), water (20 ml), brine (20 ml) and driedover magnesium sulfate. The solvent was removed in vacuo. The resultedoil was stirred with ether overnight. The suspension was filtered togive a yellowish solid. The solid was purified by HPLC (CH₃CN:Water=45:55) to give3-(4-acetylamino-1,3-dioxo-1,3-dihydro-isoindol-2-yl)-3-(3-cyclopropylmethoxy-4-difluoromethoxy-phenyl)-N,N-dimethyl-propionamideas a white solid (160 mg, 30%): mp 80-82° C.; ¹H NMR: δ(CDCl₃): δ0.33-0.39 (m, 2H), 0.60-0.68 (m, 2H), 1.21-1.30 (m, 1H), 2.25 (s, 3H),2.90 (s, 3H), 2.97 (dd, J=5 Hz, the other doublet is buried, 1H), 3.05(s, 3H), 3.87 (d, J=7.5 Hz, 2H), 3.94 (dd, J=10 Hz, the other doublet isburied, 1H), 5.83 (dd, J=5, 10 Hz, 1H), 6.69 (t, J_(H-F)=75 Hz, 1H),7.12-7.15 (m, 3H), 7.45 (d, J=7.5 Hz, 1H), 7.62 (t, J=7.5 Hz, 1H), 8.73(d, J=8.5 Hz, 1H), 9.53 (s, 1H). ¹³C NMR (CDCl₃): δ 3.8, 3.9, 10.8,25.6, 35.4, 36.1, 37.7, 51.9, 74.5, 112.6, 116.7, 120.8, 114.9, 116.0,118.5, 120.9, 123.4, 125.3, 131.9, 136.4, 138.0, 138.6, 140.7, 151.2,168.7, 169.8, 169.9, 170.6; Anal. Calcd. for C₂₆H₂₇F₂N₃O₆: C, 60.58; H,5.28; N, 8.15. Found: C, 60.23; H, 5.26; N, 8.02.

5.6 Example 6 3-(4-Acetylamino-1,3-dioxo-1,3-dihydro-isoindol-2-yl)-3-(3 cyclopropylmethoxy-4-difluoromethoxy-phenyl) -propionamide

To a solution of3-(4-acetylamino-1,3-dioxo-1,3-dihydro-isoindol-2-yl)-3-(3-cyclopropylmethoxy-4-difluoromethoxy-phenyl)-propionicacid (100 mg, 0.22 mmol) in tetrahydrofurane was addedcarbonyldiimidazole (53 mg, 0.33 mmol) at room temperature. The solutionwas stirred for 2 hours at room temperature. To the mixture was addedammonium hydroxide (0.05 ml, 0.66 mmol). The resulted mixture wasstirred at room temperature for 2 hours. Water (5 ml) was added to thereaction mixture. THF was removed in vacuo and the resulted mixture wastaken up in ethyl acetate (20 ml). The organic layer was washed withsaturated sodium bicarbonate solution (3×10 ml), water (10 ml), brine(10 ml) and was dried over magnesium sulfate. The solvent was removed invacuo. The resulted oil was purified by HPLC (acetonitrile: water=45:55)to give3-(4-acetylamino-1,3-dioxo-1,3-dihydro-isoindol-2-yl)-3-(3-cyclopropylmethoxy-4-difluoromethoxy-phenyl)-propionamideas a white solid (80 mg, 81%): mp 77-79° C.; ¹HNMR (CDCl₃): δ 0.32-0.38(m, 2H), 0.60-0.67 (m, 2H), 1.20-1.31 (m, 1H), 2.27 (s, 3H), 3.03 (dd,J=5.5, 15.5 Hz, 1H), 3.70 (dd, J=8.4, 15.5 Hz, 1H), 5.28-5.40 (m, 1H),5.74-5.80 (m, 1H), 5.79 (dd, J=5.8, 10.5 Hz, 1H), 6.60 (t, J_(H-F)=75Hz, 1H), 7.09-7.13 (m, 3H), 7.47 (d, J=7.3 Hz, 1H), 7.63 (t, J=7.5 Hz,1H), 8.75 (d, J=8.5 Hz, 1H), 9.49 (s, 1H). ¹³C NMR (CDCl₃): δ 3.8, 3.9,10.74, 25.6, 38.1, 51.9, 474.5, 112.5, 114.8, 115.8, 116.7, 118.7,120.8, 123.5, 125.6, 131.8, 136.7, 137.8, 138.1, 140.8, 151.2, 168.6,169.9, 170.5, 172.1; Anal. Calcd. for C₂₄H₂₃F₂N₃O₆+0.5 H₂O: C, 58.06%;H, 4.87; N, 8.46. Found: C, 57.77; H, 4.60; N, 8.33; 1% H₂O.

5.7 Example 7 3-(3-Cyclopropylmethoxy-4-difluoromethoxy-phenyl)-3-(1,3-dioxo-1,3-dihydro-isoindol-2-yl)-propionic acid

To a solution of3-amino-3-(3-cyclopropylmethoxy-4-difluoromethoxy-phenyl)-propionic acid(0.78 g, 2.9 mmol) and N-ethoxy-carbonyl-phthalimide (0.64 g, 2.9 mmol)in 30 ml of water and acetonitrile (1:1) was added sodium carbonate(0.33 g, 31 mmol). The mixture was stirred at room temperature for 5hours. 1N HCl was added dropwise until pH=2. The mixture was extractedwith ether (2×25 ml). The organic layer was washed with water (2×20 ml),brine (20 ml) and was dried over magnesium sulfate. The solvent wasremoved in vacuo to give3-(3-cyclopropylmethoxy-4-difluoromethoxy-phenyl)-3-(1,3-dioxo-1,3-dihydro-isoindol-2-yl)-propionicacid as a colorless oil (1.1 g, 91%). ¹HNMR (CDCl₃): δ 0.32-0.38 (m,2H), 0.60-0.67 (m, 2H), 1.17-1.28 (m, 1H), 3.33 (dd, J=6, 17 Hz, 1H),3.79 (dd, J=10, 17 Hz, 1H), 3.86 (d, J=8 Hz, 2H), 4.92 (broad, 1H), 5.74(dd, J=6, 10 Hz, 1H), 6.58 (t, J_(H-F)=75 Hz, 1H), 7.10 (s, 2H), 7.16(s, 1H), 7.69-7.74 (m, 2H), 7.78-7.83 (m, 2H).

5.8 Example 8 3-(3-Cyclopropylmethoxy-4-difluoromethoxy-phenyl)-3-(1,3-dioxo-1,3-dihydro-isoindol-2-yl)-N-hydroxy-propionamide

To a solution of3-(3-cyclopropylmethoxy-4-difluoromethoxy-phenyl)-3-(1,3-dioxo-1,3-dihydro-isoindol-2-yl)-propionicacid (1.1 g, 2.6 mmol) in tetrahydrofurane was added carbonyldiimidazole(0.73 g, 4.5 mmol) at room temperature. The solution was stirred for 2hours at room temperature. To the mixture was added hydroxylamine HClsalt (0.4 g, 2.3 mmol). The resulted mixture was stirred at roomtemperature for 4 hours. Water (20 ml) was added to the reactionmixture. THF was removed in vacuo and the resulted mixture was taken upin ethyl acetate (30 ml). The mixture was washed with saturated sodiumbicarbonate solution (3×20 ml), water (20 ml) and brine (20 ml). Theorganic layer was dried over magnesium sulfate, filtered andconcentrated in vacuo. The resulted oil was purified by silica gelchromatography (ethyl acetate:hexane=2:1) to give3-(3-cyclopropylmethoxy-4-difluoromethoxy-phenyl)-3-(1,3-dioxo-1,3-dihydro-isoindol-2-yl)-N-hydroxy-propionamideas a white solid (700 mg, 61%): mp 129-131° C.; ¹HNMR (DMSO-d₆): δ0.33-0.35 (m, 2H), 0.54-0.57 (m, 2H), 1.15-1.30 (m, 1H), 3.11 (d, J=5Hz, 2H), 3.87 (d, J=7.5 Hz, 2H), 5.71 (t, J=7.5 Hz, 1H), 6.95-6.98 (m,1H), 7.03 (t, J_(H-F)=75 Hz, 1H), 7.11-7.17 (m, 2H), 7.85 (s, 4H), 8.80(s, 1H), 10.60 (s, 1H). ¹³C NMR (CDCl₃): δ 0.7, 3.9, 3.9, 10.8, 35.7,52.0, 74.6, 112.6, 114.9, 116.7, 120.8, 120.9, 123.6, 124.2, 132.3,135.00, 137.7, 140.9, 151.3, 169.0; Anal. Calcd. for C₂₂H₂₀F₂N₂O₆: C,59.19; H, 4.52; N, 6.28. Found: C, 58.98; H, 4.41; N, 6.16.

5.9 Example 9 3-Amino-3-(3-cyclopropylmethoxy-4-difluoromethoxy-phenyl)-propionic acid methyl ester HCl salt

To a stirred suspension of3-amino-3-(3-cyclopropylmethoxy-4-difluoromethoxy-phenyl)-propionic acid(500 mg, 1.7 mmol) in methanol (10 ml) was added dropwise acetylchloride (0.3 ml, 4.3 mmol) under nitrogen atmosphere at 0° C. After theaddition, the mixture was stirred at 0° C. for 15 min and the ice bathwas removed. The mixture was stirred at room temperature overnight.Solvent was removed in vacuo. The resulted solid was stirred with ether(30 ml) for 2 hours. The suspension was filtered to give3-amino-3-(3-cyclopropylmethoxy-4-difluoromethoxy-phenyl)-propionic acidmethyl ester HCl salt as a white solid (540 mg, 90%). ¹HNMR (DMSO-d₆): δ0.30-0.36 (m, 2H), 0.54-0.61 (m, 2H), 1.22-1.32 (m, 1H), 3.01-3.10 (m,1H), 3.56 (s, 3H), 3.89 (d, J=7.5 Hz, 2H), 4.53-4.63 (m, 1H), 7.04 (d,J=7.5 Hz, 1H), 7.09 (t, J_(H-F)=75 Hz, 1H), 7.19 (d, J=7.5 Hz, 1H), 7.39(s, 1H), 8.55 (broad, 3H).

5.10 Example 103-Amino-3-(3-cyclopropylmethoxy-4-difluoromethoxy-phenyl)-propionic acidmethyl ester

To a solution of3-amino-3-(3-cyclopropylmethoxy-4-difluoromethoxy-phenyl)-propionic acidmethyl ester HCl salt (540 mg 1.5 mmol) in methylene chloride (20 ml)was added water (20 ml) and sodium carbonate (160 mg, 1.5 mmol). Themixture was stirred at room temperature for 30 min then separated inseparatory funnel. The organic layer was washed with water (20 ml),brine (20 ml) and dried over magnesium sulfate. The solvent was removedin vacuo. The resulted oil was used in next step without furtherpurification.

5.11 Example 11 3-(3-Cyclopropylmethoxy-4-difluoromethoxy-phenyl)-3-(7-nitro-1-oxo-1,3-dihydro-isoindol-2-yl)-propionic acidmethyl ester

To a solution of3-amino-3-(3-cyclopropylmethoxy-4-difluoromethoxy-phenyl)-propionic acidmethyl ester (490 mg, 1.5 mmol) and triethyl amine (0.43 ml, 3.1 mmol)in DMF (10 ml) was added 2-bromomethyl-6-nitro-benzoic acid ethyl ester(460 mg, 1.6 mmol). The mixture was heated at 90° C. under nitrogenatmosphere overnight. The solvent was removed in vacuo. The resulted oilwas extracted with ethyl acetate (50 ml) and 1N HCl (50 ml). The organiclayer was washed with water (30 ml), brine (30 ml) and dried overmagnesium sulfate. The solvent was removed in vacuo and the resulted oilwas purified by silica gel chromatography (hexane:ethyl acetate=2:1) togive3-(3-cyclopropylmethoxy-4-difluoromethoxy-phenyl)-3-(7-nitro-1-oxo-1,3-dihydro-isoindol-2-yl)-propionicacid methyl ester as a yellowish solid (550 mg, 75%). ¹HNMR (CDCl₃): δ0.31-0.37 (m, 2H), 0.59-0.67 (m, 2H), 1.22-1.28 (m, 1H), 3.09 (dd, J=7,15 Hz, 1H), 3.28 (dd, J=9, 15 Hz, 1H), 3.65 (s, 3H), 3.79-3.91 (m, 2H),4.18 (d, J=17 Hz, 1H), 4.46 (d, J=17 Hz, 1H), 5.83 (dd, J=7, 9 Hz, 1H),6.61 (t, J_(H-F)=75 Hz, 1H), 7.04-7.05 (m, 3H), 7.58-7.76 (m, 3H).

5.12 Example 12 3-(3-Cyclopropylmethoxy-4-difluoromethoxy-phenyl)-3-(7-nitro-1-oxo-1,3-dihydro-isoindol-2-yl)-propionic acid

To a suspension of3-(3-cyclopropylmethoxy-4-difluoromethoxy-phenyl)-3-(7-nitro-1-oxo-1,3-dihydro-isoindol-2-yl)-propionicacid methyl ester (550 mg, 1.2 mmol) in methanol (5 ml) was addeddropwise 10 N NaOH (0.23 ml, 2.3 mmol) at 0° C. The mixture was stirredat 0° C. for 1 hour. Then it was allowed to warm up to room temperatureand stirred for 2 hours. 4N HCl was added until pH=5. The mixture wasextracted with methylene chloride (30 ml) and 1N HCl (30 ml). Theorganic layer was washed with water (2×30 ml), brine (30 ml) and driedover magnesium sulfate. The solvent was removed in vacuo to give3-(3-cyclopropylmethoxy-4-difluoromethoxy-phenyl)-3-(7-nitro-1-oxo-1,3-dihydro-isoindol-2-yl)-propionicacid as a yellow solid (480 mg, 90%). ¹HNMR (CDCl₃): δ 0.31-0.37 (m,2H), 0.59-0.67 (m, 2H), 1.22-1.28 (m, 1H), 3.11 (dd, J=6.5, 15 Hz, 1H),3.33 (dd, J=9, 15 Hz, 1H), 3.78-3.87 (m, 2H), 4.18 (d, J=17.5 Hz, 1H),4.46 (d, J=17.5 Hz, 1H), 5.83 (dd, J=6.5, 9 Hz, 1H), 6.61 (t, J_(H-F)=75Hz, 1H), 6.91-7.17 (m, 3H), 7.59-7.76 (m, 3H).

5.13 Example 13 3-(3-Cyclopropylmethoxy-4-difluoromethoxy-phenyl-3-(7-nitro-1-oxo-1,3-dihydro-isoindol-2-yl)-)-N,N-dimethyl-propionamide

To a solution of3-(3-cyclopropylmethoxy-4-difluoromethoxy-phenyl)-3-(7-nitro-1-oxo-1,3-dihydro-isoindol-2-yl)-propionicacid (480 mg, 1.0 mmol) in tetrahydrofurane (5 ml) was addedcarbonyldiimidazole (250 mg, 1.6 mmol) at room temperature. The solutionwas stirred at room temperature for 2 hours. To the mixture was addeddimethylamine (2.0M in THF, 1.0 ml, 2.0 mmol). The resulted mixture wasstirred at room temperature for 3 hours. Water (20 ml) was added to thereaction mixture. THF was removed in vacuo and the resulted mixture wastaken up in ethyl acetate (30 ml). The mixture was washed with saturatedsodium bicarbonate solution (3×20 ml), water (20 ml) and brine (20 ml).The organic layer was dried over magnesium sulfate, filtered andconcentrated in vacuo. The resulted oil was stirred with etherovernight. The suspension was filtered to give3-(3-cyclopropylmethoxy-4-difluoromethoxy-phenyl-3-(7-nitro-1-oxo-1,3-dihydro-isoindol-2-yl)-)-N,N-dimethyl-propionamideas a yellow solid (330 mg, 65%). ¹HNMR (CDCl₃): δ 0.32-0.38 (m, 2H),0.60-0.67 (m, 2H), 1.15-1.35 (m, 1H), 2.89 (s, 3H), 3.00 (dd, J=5, 15Hz, 1H), 3.10 (s, 3H), 3.79 (dd, J=10, 15 Hz, 1H), 3.85-3.89 (m, 2H),4.44 (d, J=17.5 Hz, 1H), 4.57 (d, J=17.5 Hz, 1H), 5.47 (dd, J=5, 10 Hz,1H), 6.61 (t, J_(H-F)=75 Hz, 1H), 6.98-7.02 (m, 1H), 7.12-7.20 (m, 2H),7.56-7.72 (m, 3H).

5.14 Example 143-(7-Amino-1-oxo-1,3-dihydro-isoindol-2-yl)-3-(3-cyclopropylmethoxy-4-difluoromethoxy-phenyl)-N,N-dimethyl-propionamide

To a solution of3-(3-cyclopropylmethoxy-4-difluoromethoxy-phenyl)-3-(7-nitro-1-oxo-1,3-dihydro-isoindol-2-yl)-N,N-dimethyl-propionamide(330 mg, 0.67 mmol) in ethyl acetate (100 ml) was added 10% Pd on carbon(100 mg). The suspension was shaked under about 50 psi hydrogenatmosphere at room temperature overnight. The suspension was filteredthrough a pad of celite. The solvent was removed and the resulted oilwas purified by HPLC (CH₃CN:water=40:60) to give3-(7-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-3-(3-cyclopropylmethoxy-4-difluoromethoxy-phenyl)-N,N-dimethyl-propionamideas a white solid (300 mg, 65%): mp 70-72° C.; ¹HNMR (CDCl₃): δ 0.31-0.37(m, 2H), 0.58-0.65 (m, 2H), 1.21-1.28 (m, 1H), 2.90 (s, 3H), 3.05 (dd,J=6.3, 15.8 Hz, 1H), 3.09 (s, 3H), 3.52 (dd, J=8.5, 15.3 Hz, 1H), 3.85(d, J=7 Hz, 2H), 4.20 (d, J=16.8 Hz, 1H), 4.35 (d, J=16.8 Hz, 1H), 5.20(broad, 2H), 5.56 (dd, J=6.3, 8.5 Hz, 1H), 6.52-6.63 (m, 2H), 6.59 (t,J_(H-F)=75 Hz, 1H), 6.90-6.98 (m, 1H), 7.09-7.26 (m, 3H); ¹³C NMR(CDCl₃): δ 10.8, 36.2, 37.0, 38.1, 49.8, 54.3, 114.0, 114.9, 116.3,116.9, 120.1, 121.0, 123.3, 133.4, 139.4, 140.5, 143.4, 146.6, 151.3,170.4, 170.9; Anal. Calcd. for C₂₄H₂₇F₂N₃O₄+0.18 H₂O: C, 62.30; H, 5.96;N, 9.08; H₂O 0.7. Found: C, 62.30; H, 5.89; N, 9.02; H₂O, 0.7.

5.15 Example 15 4-Difluoromethoxy-3-ethoxy-benzaldehyde

To a dioxane solution (1000 ml) of 3-ethoxy-4-hydroxy benzaldehyde (100g, 0.61 mol) was added phase transfer catalyst benzyl trimethyl ammoniumchloride (3.4 g, 0.018 mole) followed by aqueous solution of NaOH (72 g,in 72 ml of water, 1.8 mole). The solution became cloudy after addingNaOH. Into the suspension was bubbled gas difluorochloro methane (100 gin steel cylinder) with vigorous stirring. The system was closed duringthe bubbling with careful control of the release of the gas. Theresulted suspension was stirred overnight at room temperature. Thesuspension was poured into 1000 ml of crushed ice and the mixture wasextracted with EtOAc (300 ml×4). The combined organic layer was driedover MgSO₄, filtered and concentrated. The resulted brown oil waspurified by silica gel chromatography (eluent is 100% toluene) to give40 g of product as colorless oil (31%).

5.16 Example 16 3-Amino-3-(4-difluoromethoxy-3-ethoxy-phenyl) -propionicacid

4-Difluoromethoxy-3-ethoxy-benzaldehyde (10 g, 0.046 mole) was added to95% ethanol (100 ml) followed by NH₄OAc (7.1 g, 0.092 mol). The mixturewas heated to 40° C. for an hour then malonic acid (4.8 g, 0.046 mole)was added. The mixture was stirred at 76° C. overnight. A yellowsuspension was obtained. It was filtered and the solid was rinsed withethanol (10 ml) to give3-amino-3-(4-difluoromethoxy-3-ethoxy-phenyl)-propionic mid as whitesolid (9.7 g, 77%). ¹HNMR (DMSO-d₆): δ 1.41 (t, J=7.5 Hz, 3H), 3.32 (d,J=7 Hz, 1H), 2.67 (s, 1H), 4.09 (q, J=7.5 Hz, 2H), 4.20-4.24 (m, 1H),6.97 (d, J=8.0 Hz, 1H), 7.03 (t, J_(H-F)=75 Hz, 1H), 7.13 (d, J=8.0 Hz,1H), 7.23 (s, 1H).

5.17 Example 17 3-Amino-3-(4-difluoromethoxy-3-ethoxy-phenyl) -propionicacid methyl ester

3-Amino-3-(4-difluoromethoxy-3-ethoxy-phenyl)-propionic acid (7.6 g, 28mmol) was suspended in methanol (50 ml). To the mixture was addeddropwise acetyl chloride (4.9 ml, 69 mmol) at room temperature. Afterthe addition, the mixture turned into a clear solution and was stirredat room temperature for 5 hours. HPLC showed all the starting materialwas gone. The solvent was removed in vacuo and the resulted solid wasstirred with ether (10 ml) for 1 hour. The suspension was filtered and 9g of solid was obtained. The solid was then suspended in methylenechloride (100 ml). To it was added Na₂CO₃ aqueous solution (3.3 g in 50ml of water). The mixture was stirred for 30 min and separated. Theorganic layer was washed with water (50 ml×2), brine (50 ml), dried overNa₂SO₄ and concentrated to give3-amino-3-(4-difluoromethoxy-3-ethoxy-phenyl)-propionic acid methylester (7.3 g, 92%). ¹HNMR (CDCl₃): δ 1.41 (t, J=7.5 Hz, 3H), 2.62-2.64(m, 1H), 3.69 (s, 3H), 4.10 (q, J=7.5 Hz, 2H), 4.37-4.32 (m, 1H), 6.55(t, J_(H-F)=75 Hz, 1H). 6.89 (dd, J=1.8, 8 Hz, 1H), 7.01 (d, J=1.8 Hz,1H), 7.11 (d, J=8 Hz, 1H).

5.18 Example 183-(4-Difluoromethoxy-3-ethoxy-phenyl)-3-(7-nitro-1-oxo-1,3-dihydro-isoindol-2-yl)-propionicacid methyl ester

3-(4-Difluoromethoxy-3-ethoxy-phenyl)-3-(7-nitro-1-oxo-1,3-dihydro-isoindol-2-yl)-propionicacid methyl ester was prepared by the procedure of example 11 from3-amino-3-(4-difluoromethoxy-3-ethoxy-phenyl)-propionic acid methylester (7.3 g, 0.025 mol), 2-bromomethyl-6-nitro-benzoic acid ethyl ester(7.6 g, 0.028 mol) and triethyl amine (7 ml, 0.05 mol) in DMF (50 ml) toprovide3-(4-difluoromethoxy-3-ethoxy-phenyl)-3-(7-nitro-1-oxo-1,3-dihydro-isoindol-2-yl)-propionicacid methyl ester as yellow oil (9.1 g, 81% yield). ¹HNMR (CDCl₃): δ1.42 (t, J=7.5 Hz, 3H, OCH₂CH₃), 3.09 (dd, J=6.8, 15 Hz, 2H, CH₂CO),3.29 (dd, J=6.8, 15 Hz, 1H, CH₂CO), 3.65 (s, 3H, OCH₃), 4.10 (q, J=7.5Hz, 2H, OCH₂CH₃), 4.26 (d, J=17 Hz, 2H, CH2N), 4.46 (d, J=17 Hz, 2H,CH2N), 5.80-5.86 (m, 1H, CHN), 6.55 (t, J_(H-F)=75 Hz, 1H, OCF₂H), 6.92(dd, J=1.8, 8.2 Hz, 1H, Ar), 7.06 (d, J=1.8 Hz, 1H, Ar), 7.14 (d,J=8.25, 1H, Ar), 7.58-7.76 (m, 3H, Ar).

5.19 Example 193-(7-Amino-1-oxo-1,3-dihydro-isoindol-2-yl)-3-(4-difluoromethoxy-3-ethoxy-phenyl)-propionicacid methyl ester

3-(7-Amino-1-oxo-1,3-dihydro-isoindol-2-yl)-3-(4-difluoromethoxy-3-ethoxy-phenyl)-propionicacid methyl ester was prepared by the procedure of example 14 from3-(4-difluoromethoxy-3-ethoxy-phenyl)-3-(7-nitro-1-oxo-1,3-dihydro-isoindol-2-yl)-propionicacid methyl ester (9.1 g, 0.02 mol) and palladium on carbon underhydrogen pressure (50 psi). The product was used in the next stepwithout further purification.

5.20 Example 203-[7-(Cyclopropanecarbonyl-amino)-1-oxo-1,3-dihydro-isoindol-2-yl]-3-(4-difluoromethoxy-3-ethoxy-phenyl)-propionic acid methyl ester

To the THF solution of3-(7-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-3-(4-difluoromethoxy-3-ethoxy-phenyl)-propionicacid methyl ester (2.8 g, 7 mmol) was added cyclopropanyl carbonylchloride (0.72 ml, 8 mmol) dropwise. The reaction mixture was thenheated to reflux for 1 hour. The solvent was removed under vacuo. Themixture was dissolved in EtOAc (50 ml) and washed with water (50 ml×2).The organic layer was dried over Na₂SO₄ and concentrated under vacuo.The resulted solid was stirred with ether (20 ml) for 1 hour andfiltered to give a total of 2.5 g (77% yield) white solid which was usedin the next step without further purification.

5.21 Example 213-(7-Acetylamino-1-oxo-1,3-dihydro-isoindol-2-yl)-3-(4-difluoromethoxy-3-ethoxy-phenyl)-propionicacid methyl ester

3-(7-Acetylamino-1-oxo-1,3-dihydro-isoindol-2-yl)-3-(4-difluoromethoxy-3-ethoxy-phenyl)-propionicacid methyl ester was prepared by the procedure of example 20 from3-(7-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-3-(4-difluoromethoxy-3-ethoxy-phenyl)-propionicacid methyl ester (2.8 g, 7 mmol) and acetyl chloride (0.57 ml, 8 mmol)in THF (20 ml) to afford the title compound as white solid (2.5 g, 81%).The product was used in the next step without further purification.

5.22 Example 223-(7-Acetylamino-1-oxo-1,3-dihydro-isoindol-2-yl)-3-(4-difluoromethoxy-3-ethoxy-phenyl)-propionicacid

To the THF solution of3-(7-acetylamino-1-oxo-1,3-dihydro-isoindol-2-yl)-3-(4-difluoromethoxy-3-ethoxy-phenyl)-propionicacid methyl ester (2.5 g, 5.4 mmol) was added dropwise 10N NaOH (1 ml,10 mmol) and was stirred at room temperature overnight. The resultedsuspension was filtered and the solid was dissolved in water (20 ml).The aqueous solution was acidified to pH=4 to obtain a milky suspension.The suspension was extracted with EtOAc (50 ml×3). The organic layer waswashed with water (50 ml×2), brine (30 ml), dried over Na₂SO₄ andconcentrated to give white solid (2.3 g, 96% yield): mp 105-107° C.;¹HNMR (CDCl₃): δ 1.42 (J=7.5 Hz, 3H, OCH₂CH₃), 2.22 (s, 3H), 3.11 (dd,J=5, 12 Hz, 1H, CH₂), 3.25 (dd, J=10, 15 Hz, 1H, CH₂), 4.08 (q, J=7.5Hz, 2H, OCH₂CH₃), 4.12 (d, J=17.5 Hz, 1H, CH₂N), 4.38 (d, J=17.5 Hz, 1H,CH₂N), 5.81 (m, 1H, CHN), 6.55 (t, J_(H-F)=75 Hz, 1H, CF₂H), 6.96 (m,3H, Ar), 7.13 (d, J=7.5 Hz, 1H, Ar), 7.45 (m, 1H, Ar), 8.43 (d, J=7.5Hz, 1H, Ar), 10.25 (s, 1H, NHCO). ¹³CNMR (CDCl₃): δ14.6, 24.8, 36.7,46.8, 51.7, 64.9, 111.8, 111.3, 115.9, 117.1, 117.3, 118.0, 118.9,120.1, 122.8, 133.5, 136.7, 137.8, 140.1, 141.2, 150.9, 169.47, 169.49,173.5: Anal. Calcd. for C₂₂H₂₂F₂N₂O₆+0.22H₂O: C, 58.41; H, 5.00; N,6.19. Found: C, 58.06, H, 4.92, N, 5.90.

5.23 Example 233-[7-(Cyclopropanecarbonyl-amino)-1-oxo-1,3-dihydro-isoindol-2-yl]-3-(4-difluoromethoxy-3-ethoxy-phenyl)-propionic acid

3-[7-(Cyclopropanecarbonyl-amino)-1-oxo-1,3-dihydro-isoindol-2-yl]-3-(4-difluoromethoxy-3-ethoxy-phenyl)-propionicacid was prepared by the procedure of Example 22 from3-(7-cyclopropanecarbonyl-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-3-(4-difluoromethoxy-3-ethoxy-phenyl)-propionicacid methyl ester (2.47 g, 5 mmol) and NaOH (10N, 1 ml, 10 mmol) in THF(50 ml) to give3-[7-(cyclopropanecarbonyl-amino)-1-oxo-1,3-dihydro-isoindol-2-yl]-3-(4-difluoromethoxy-3-ethoxy-phenyl)-propionicacid as white solid (2.5 g) and used in the next step without furtherpurification.

5.24 Example 24 Cyclopropanecarboxylic acid{2-[2-carbamoyl-1-(4-difluoromethoxy-3-ethoxy-phenyl)-ethyl]-3-oxo-2,3-dihydro-1H-isoindol-4-yl}-amide

Cyclopropanecarboxylic acid{2-[2-carbamoyl-1-(4-difluoromethoxy-3-ethoxy-phenyl)-ethyl]-3-oxo-2,3-dihydro-1H-isoindol-4-yl}-amidewas prepared by the procedure of example 6 from3-[7-(cyclopropanecarbonyl-amino)-1-oxo-1,3-dihydro-isoindol-2-yl]-3-(4-difluoromethoxy-3-ethoxy-phenyl)-propionicacid (0.8 g, 1.7 mmol), CDI (0.41 g, 2.5 mmol) and NH₄OH (0.25 ml. 3.4mmol) in THF (30 ml) to give cyclopropanecarboxylic acid{2-[2-carbamoyl-1-(4-difluoromethoxy-3-ethoxy-phenyl)-ethyl]-3-oxo-2,3-dihydro-1H-isoindol-4-yl}-amideas white solid (0.56 g, 70%): mp, 179-181° C.; ¹HNMR (CDCl₃): δ 0.92 (m,2H, c-CH₂), 1.44 (t, J=6.8 Hz, 3H, OCH₂CH₃), 1.68 (m, 1H, c-CH), 2.99(dd, J=5, 15 Hz, 1H, CH₂), 3.44 (dd, J=10, 15 Hz, 1H, CH₂), 4.08 (q,J=7.0 Hz, 2H, OCH₂CH₃), 4.20 (d, J=17.5 Hz, 1H, CH₂N), 4.40 (d, J=17.5Hz, 1H, CH₂N), 5.42 (broad, 1H, NH₂), 5.58 (m, 1H, CHN), 6.07 (broad,1H), 6.56 (t, J_(H-F)=75 Hz, 1H, CF₂H), 6.98 (m, 3H, Ar), 7.15 (d, J=7.5Hz, 1H, Ar), 7.46 (t, J=7.5 Hz, 1H, Ar), 8.45 (d, J=8 Hz, 1H, Ar), 10.49(s, 1H, NHCO). ¹³CNMR (CDCl₃):

8.31, 14.6, 16.2, 38.9, 48.6, 53.9, 64.8, 101.57, 102.3, 113.2, 116.7,117.3, 117.8, 119.2, 122.9, 113.5, 137.3, 138.1, 141.4, 141.2, 150.9,170.9, 171.4, 172.7; Anal. Calcd. for C₂₄H₂₅F₂N₃O₅: C, 60.88; H, 5.32;N, 8.87. Found: C, 60.82; H, 5.11; N, 8.80.

5.25 Example 25 Cyclopropanecarboxylic acid{2-[1-(4-difluoromethoxy-3-ethoxy-phenyl)-2-dimethylcarbamoyl-ethyl]-3-oxo-2,3-dihydro-1H-isoindol-4-yl}-amide

Cyclopropanecarboxylic acid{2-[1-(4-difluoromethoxy-3-ethoxy-phenyl)-2-dimethylcarbamoyl-ethyl]-3-oxo-2,3-dihydro-1H-isoindol-4-yl}-amidewas prepared by the procedure of example 13 from3-[7-(cyclopropanecarbonyl-amino)-1-oxo-1,3-dihydro-isoindol-2-yl]-3-(4-difluoromethoxy-3-ethoxy-phenyl)-propionicacid (0.8 g, 1.7 mmol), CDI (0.41 g, 2.5 mmol) and dimethylamine (1.7ml, 3.4 mmol) in THF (30 ml) to give cyclopropanecarboxylic acid{2-[1-(4-difluoromethoxy-3-ethoxy-phenyl)-2-dimethylcarbamoyl-ethyl]-3-oxo-2,3-dihydro-1H-isoindol-4-yl}-amideas white solid (0.4 g, 50% yield): mp: 135-137° C.; ¹HNMR (CDCl₃): δ0.89 (m, 2H, c-CH₂), 1.43 (t, J=7.0 Hz, 3H, OCH₂CH₃), 1.66 (m, 1H,c-CH). 2.93 (s, 3H, CH₃CO), 3.04 (dd, J=5, 15 Hz, 1H, CH₂), 3.10 (s, 3H,NCH₃), 3.60 (dd, J=10, 15 Hz, 1H, CH₂), 4.09 (q, J=7.0 Hz, 2H, OCH₂CH₃),4.33 (d, J=17.5 Hz, 1H, CH₂N), 4.44 (d, J=17.5 Hz, 1H, CH₂N), 5.60 (m,1H, CHN), 6.55 (t, J_(H-F)=75 Hz, 1H, CF₂H), 7.07 (m, 4H, Ar), 7.43 (t,J=7.5 Hz, 1H, Ar), 8.42 (d, J=7.5 Hz, 1H, Ar), 10.57 (s, 1H, NHCO).¹³CNMR (CDCl₃): δ 8.2, 14.6, 16.1, 35.6, 36.1, 37.3, 49.2, 54.0, 58.6,64.7, 111.9, 113.5, 116.1, 116.7, 117.5, 117.6, 117.8, 119.2, 120.2,122.7, 133.1, 138.0, 138.2, 139.7, 141.5, 159.7, 169.4, 169.7, 172.6;Anal. Calcd. for C₂₆H₂₉F₂N₃O₅: C, 62.27; H, 5.83; N, 8.38. Found: C,62.07; H, 5.65; N, 8.28.

5.26 Example 26 Cyclopropanecarboxylic acid{2-[1-(4-difluoromethoxy-3-ethoxy-phenyl)-2-hydroxycarbamoyl-ethyl]-3-oxo-2,3-dihydro-1H-isoindol-4-yl}-amide

Cyclopropanecarboxylic acid{2-[1-(4-difluoromethoxy-3-ethoxy-phenyl)-2-hydroxycarbamoyl-ethyl]-3-oxo-2,3-dihydro-1H-isoindol-4-yl}-amidewas prepared by the procedure of example 8 from3-[7-(cyclopropanecarbonyl-amino)-1-oxo-1,3-dihydro-isoindol-2-yl]-3-(4-difluoromethoxy-3-ethoxy-phenyl)-propionicacid (0.8 g, 1.7 mmol), CDI (0.41 g, 2.5 mmol) and hydroxylaminehydrochloride (0.23 g, 3.6 mmol) in THF (30 ml) to givecyclopropanecarboxylic acid{2-[1-(4-difluoromethoxy-3-ethoxy-phenyl)-2-hydroxycarbamoyl-ethyl]-3-oxo-2,3-dihydro-1H-isoindol-4-yl}-amideas white solid (0.60 g, 80% yield): mp 116-118° C.; ¹HNMR (CDCl₃): δ0.82 (m, 2H, c-CH₂), 1.02 (m, 2H, c-CH₂), 1.36 (t, J=6.8 Hz, 3H,OCH₂CH₃), 1.58 (m, 1H, c-CH), 2.86 (dd, J=5, 15 Hz, 1H, CH₂), 3.15 (dd,J=10, 15 Hz, 1H, CH₂), 3.30 (broad, 1H, NHOH), 4.01 (q, J=7.0 Hz, 2H,OCH₂CH₃), 4.02 (d, J=17.5 Hz, 1H, CH₂N), 4.34 (d, J=17.5 Hz, 1H, CH₂N),5.42 (broad, 1H, NH₂), 5.71 (m, 1H, CHN), 6.07 (broad, 1H, NHOH), 6.53(t, J_(H-F)=75 Hz, 1H, CF₂H), 6.88 (m, 3H, Ar), 7.07 (d, J=7.5 Hz, 1H,Ar), 7.30 (t, J=7.5 Hz, 1H, Ar), 8.28 (d, J=8 Hz, 1H, Ar), 10.35 (s, 1H,NHCO). ¹³CNMR (CDCl₃):

8.27, 8.39, 14.5, 16.0, 35.4, 47.3, 52.4, 64.8, 111.8, 113.3, 115.9,116.9, 117.0, 118.9, 120.0, 122.6, 133.3, 136.6, 137.6, 140.0, 141.5,150.8, 167.5, 169.7, 172.8; Anal. Calcd. for C24H25F2N3O6+0.34 H₂O: C,58.16; H, 5.22; N, 8.48. Found: C, 58.25; H, 5.37; N, 8.58, 1.25 H₂O

5.27 Example 273-(7-Acetylamino-1-oxo-1,3-dihydro-isoindol-2-yl)-3-(4-difluoromethoxy-3-ethoxy-phenyl)-propionamide

3-(7-Acetylamino-1-oxo-1,3-dihydro-isoindol-2-yl)-3-(4-difluoromethoxy-3-ethoxy-phenyl)-propionamidewas prepared by the procedure of example 6 from3-[7-(acetylamino)-1-oxo-1,3-dihydro-isoindol-2-yl]-3-(4-difluoromethoxy-3-ethoxy-phenyl)-propionicacid (0.6 g, 1.3 mmol), CDI (0.3 g, 2 mmol) and NH₄OH (0.20 ml, 2.6mmol) in THF (30 ml) to give3-(7-acetylamino-1-oxo-1,3-dihydro-isoindol-2-yl)-3-(4-difluoromethoxy-3-ethoxy-phenyl)-propionamideas white solid (0.43 g, 72%): mp 129-131° C.; ¹HNMR (CDCl₃): δ 1.44 (t,J=7.0 Hz, 3H, OCH₂CH₃), 2.25 (s, 3H, CH₃CO), 2.97 (dd, J=5, 15 Hz, 1H,CH₂), 3.42 (dd, J=10, 15 Hz, 1H, CH₂), 4.08 (q, J=7.0 Hz, 2H, OCH₂CH₃),4.20 (d, J=17.5 Hz, 1H, CH₂N), 4.40 (d, J=17.5 Hz, 1H, CH₂N), 5.40(broad, 1H, NH₂), 5.56 (m, 1H, CHN), 6.01 (broad, 1H), 6.56 (t,J_(H-F)=75 Hz, 1H, CF₂H), 6.95 (m, 3H, Ar), 7.15 (d, J=7.5 Hz, 1H, Ar),7.47 (t, J=7.5 Hz, 1H, Ar), 8.47 (d, J=7.5 Hz, 1H, Ar), 10.25 (s, 1H,NHCO). ¹³CNMR (DMSO-d₆): δ 3.2, 14.4, 16.2, 24.4, 37.7, 46.7, 51.7,64.2, 93.9, 113.0, 116.6, 117.2, 117.5, 119.2, 121.3, 132.8, 137.0,138.2, 139.0, 142.2, 149.8, 168.0, 168.5, 171.0; Anal. Calcd. forC₂₂H₂₃F₂N₃O₅: C, 59.06; H, 5.18; N, 9.39. Found: C, 58.48; H, 5.12; N,9.65.

5.28 Example 283-(7-Acetylamino-1-oxo-1,3-dihydro-isoindol-2-yl)-3-(4-difluoromethoxy-3-ethoxy-phenyl)-N,N-dimethyl-propionamide

3-(7-Acetylamino-1-oxo-1,3-dihydro-isoindol-2-yl)-3-(4-difluoromethoxy-3-ethoxy-phenyl)-N,N-dimethyl-propionamidewas prepared by the procedure of example 13 from3-(7-acetylamino-1-oxo-1,3-dihydro-isoindol-2-yl)-3-(4-difluoromethoxy-3-ethoxy-phenyl)-propionicacid (0.1 g, 0.22 mmol), CDI (0.05 g, 0.33 mmol) and dimethylamine (0.33ml, 0.66 mmol) in THF (10 ml) to give as3-(7-acetylamino-1-oxo-1,3-dihydro-isoindol-2-yl)-3-(4-difluoromethoxy-3-ethoxy-phenyl)-N,N-dimethyl-propionamidewhite solid (0.05 g, 50% yield): mp 141-143° C.; ¹HNMR (CDCl₃): δ 1.44(t, J=7.0 Hz, 3H, OCH₂CH₃), 2.25 (s, 3H, CH₃CO), 2.93 (s, 3H, NCH₃),3.04 (dd, J=5, 15 Hz, 1H, CH₂), 3.10 (s, 3H, NCH₃), 3.60 (dd, J=10, 15Hz, 1H, CH₂), 4.09 (q, J=7.0 Hz, 2H, OCH₂CH₃), 4.33 (d, J=18 Hz, 1H,CH₂N), 4.44 (d, J=18 Hz, 1H, CH₂N), 5.59 (m, 1H, CHN), 6.55 (t,J_(H-F)=75 Hz, 1H, CF₂H), 7.04 (m, 4H, Ar), 7.45 (t, J=7.5 Hz, 1H, Ar),8.45 (d, J=7.5 Hz, 1H, Ar), 10.35 (s, 1H, NHCO). ¹³CNMR (DMSO-d₆):

14.5, 24.8, 35.5, 35.9, 37.2, 49.0, 53.8, 64.7, 111.9, 113.4, 113.5,116.0, 116.8, 117.5, 117.8, 119.1, 119.1, 120.1, 122.6, 133.0, 137.7,138.2, 139.7, 138.2, 139.6, 139.7, 141.5, 150.6, 168.9, 169.2, 169.5;Anal. Calcd. for C₂₄H₂₇F₂N₃O₅: C, 60.62; H, 5.72; N, 8.84. Found: C,59.90; H, 5.46; N, 8.66.

5.29 Example 293-(7-Acetylamino-1-oxo-1,3-dihydro-isoindol-2-yl)-3-(4-difluoromethoxy-3-ethoxy-phenyl)-N-hydroxy-propionamide

3-(7-Acetylamino-1-oxo-1,3-dihydro-isoindol-2-yl)-3-(4-difluoromethoxy-3-ethoxy-phenyl)-N-hydroxy-propionamidewas prepared by the procedure of example 8 from3-(7-acetylamino-1-oxo-1,3-dihydro-isoindol-2-yl)-3-(4-difluoromethoxy-3-ethoxy-phenyl)-propionicacid (0.6 g, 1.3 mmol), CDI (0.32 g, 2.0 mmol) and hydroxylaminehydrocloride (0.19 g, 2.6 mmol) in THF (30 ml) to give3-(7-acetylamino-1-oxo-1,3-dihydro-isoindol-2-yl)-3-(4-difluoromethoxy-3-ethoxy-phenyl)-N-hydroxy-propionamideas white solid (0.22 g, 40% yield): mp 121-123° C.; ¹HNMR (DMSO-d₆):

1.34 (t, J=7.5 Hz, 3H, OCH₂CH₃), 2.16 (s, 3H, CH₃), 2.89 (d, J=7.5 Hz,2H, CH₂), 4.10 (q, J=7.0 Hz, 2H, OCH₂CH₃), 4.24 (d, J=17.5 Hz, 1H,CH₂N), 4.59 (d, J=17.5 Hz, 1H, CH₂N), 5.69 (m, 1H, CHN), 6.93 (m, 1H,Ar), 7.03 (t, J_(H-F)=75 Hz, 1H, CF₂H), 7.15 (m, 3H, Ar), 7.52 (t, J=7.5Hz, 1H, Ar), 8.26 (d, J=8 Hz, 1H, Ar), 8.85 (s, 1H, NHOH), 10.26 (s, 1H,NHOH), 10.35 (s, 1H, NHCO). ¹³CNMR (DMSO-d₆): δ14.4, 24.4, 34.5, 51.5,64.2, 93.9, 113.0, 116.6, 117.3, 117.5, 120.7, 121.3, 122.5, 132.8,134.0, 137.0, 137.8, 142.2, 149.8, 165.7, 168.0, 168.5; Anal. Calcd. forC₂₂H₂₃F₂N₃O₆+0.13 H₂O: C, 56.73; H, 5.03; N, 9.02. Found: C, 56.35; H,4.89; N, 8.75; H₂O 0.34.

5.30 Example 303-(4-Acetylamino-1,3-dioxo-1,3-dihydro-isoindol-2-yl)-3-(4-difluoromethoxy-3-ethoxy-phenyl)-propionicacid

3-(4-Acetylamino-1,3-dioxo-1,3-dihydro-isoindol-2-yl)-3-(4-difluoromethoxy-3-ethoxy-phenyl)-propionicacid was prepared by the procedure of Example 4 from3-amino-3-(4-difluoromethoxy-3-ethoxy-phenyl)-propionic acid (2.7 g, 13mmol), 3-acetamido-phthalic anhydride (4.0 g, 14.5 mmol) and sodiumacetate (1.2 g, 14.5 mmol) in acetic acid (50 ml) to give3-(4-acetylamino-1,3-dioxo-1,3-dihydro-isoindol-2-yl)-3-(4-difluoromethoxy-3-ethoxy-phenyl)-propionicacid (2.6 g, 45%) as yellowish solid. mp 89-91° C.; ¹HNMR (CDCl₃):

1.42 (t, J=7.5 Hz, 3H, OCH₂CH₃), 2.51 (s, 3H, CH₃CO), 3.19 (dd, J=5.8,15.5 Hz, 1H, CH₂), 3.79 (dd, J=8.4, 15.5 Hz, 1H, CH₂), 4.08 (q, J=7.5Hz, 2H, OCH2CH3), 5.65-5.71 (m, 1H, CHN), 6.52 (t, J_(H-F)=75 Hz, 1H,CF₂H), 7.03-7.11 (m, 3H, Ar), 7.45 (d, J=7.5 Hz, 1H, Ar), 7.63 (t, J=7.5Hz, 1H, Ar), 8.72 (d, J=8.3 Hz, 1H, Ar), 9.46 (s, 1H, NHCO). ¹³C NMR(CDCl₃):

14.6, 24.8, 35.6, 50.3, 64.7, 98.4, 99.1, 111.8, 113.5, 115.2, 115.9,118.2, 120.1, 120.2, 122.7, 125.0, 131.0, 136.0, 136.5, 137.4, 138.1,140.0, 140.1, 140.2, 150.6, 167.4, 169.5, 169.7, 175.1; Anal. Calcd. forC₂₂H₂₀F₂N₂O₇+0.17 H₂O: C, 56.77; H, 4.40; N, 6.06; H₂O 0.66. Found: C,56.60; H, 4.43; N, 6.02; H₂O 0.66.

5.31 Example 313-(4-Acetylamino-1,3-dioxo-1,3-dihydro-isoindol-2-yl)-3-(4-difluoromethoxy-3-ethoxy-phenyl)-propionamide

3-(4-Acetylamino-1,3-dioxo-1,3-dihydro-isoindol-2-yl)-3-(4-difluoromethoxy-3-ethoxy-phenyl)-propionamidewas prepared by the procedure of Example 6 from3-(4-acetylamino-1,3-dioxo-1,3-dihydro-isoindol-2-yl)-3-(4-difluoromethoxy-3-ethoxy-phenyl)-propionicacid (0.5 g, 1.1 mmol), CDI (0.19 g, 1.2 mmol) and NH₄OH (0.1 ml, 1.2mmol) in THF (30 ml) to give3-(4-acetylamino-1,3-dioxo-1,3-dihydro-isoindol-2-yl)-3-(4-difluoromethoxy-3-ethoxy-phenyl)-propionamideas white solid (0.40 g, 80%): mp 178-180° C.; ¹HNMR (CDCl₃): δ1.44 (t,J=7.0 Hz, 3H, OCH₂CH₃), 2.26 (s, 3H, CH₃CO), 3.03 (dd, J=5, 15 Hz, 1H,CH₂), 3.70 (dd, J=10, 15 Hz, 1H, CH₂), 4.09 (q, J=7.0 Hz, 2H, OCH₂CH₃),5.38 (broad, 1H, NH₂), 5.61 (broad, 1H, NH₂), 5.76-5.83 (m, 1H, CHN),6.83 (t, J_(H-F)=75 Hz, 1H, CF₂H), 7.06-7.14 (m, 3H, Ar), 7.46 (d, J=7.5Hz, 1H, Ar), 7.65 (t, J=7.5 Hz, 1H, Ar), 7.74 (d, J=7.5 Hz, 1H, Ar),9.45 (s, 1H, NHCO). ¹³CNMR (DMSO-d₆):

14.4, 24.2, 36.7, 50.0, 64.2, 93.9, 112.5, 113.2, 116.7, 116.7, 118.0,119.4, 121.2, 125.9, 131.4, 135.8, 136.4, 137.6, 139.0, 149.7, 167.2,168.2, 169.2, 171.0; Anal Calcd for C₂₂H₂₁F₂N₃O₆: C, 57.27; H, 4.59; N,9.11. Found: C, 57.04; H, 4.41; N, 8.93.

5.32 Example 323-(4-Acetylamino-1,3-dioxo-1,3-dihydro-isoindol-2-yl)-3-(4-difluoromethoxy-3-ethoxy-phenyl)-N,N-dimethyl-propionamide

3-(4-Acetylamino-1,3-dioxo-1,3-dihydro-isoindol-2-yl)-3-(4-difluoromethoxy-3-ethoxy-phenyl)-N,N-dimethyl-propionamidewas prepared by the procedure of example 13 from3-(4-acetylamino-1,3-dioxo-1,3-dihydro-isoindol-2-yl)-3-(4-difluoromethoxy-3-ethoxy-phenyl)-propionicacid (0.5 g, 1.1 mmol), CDI (0.2 g, 0.1.3 mmol) and dimethylamine (2N inTHF, 0.7 ml, 1.4 mmol) in THF (10 ml) to give3-(4-acetylamino-1,3-dioxo-1,3-dihydro-isoindol-2-yl)-3-(4-difluoromethoxy-3-ethoxy-phenyl)-N,N-dimethyl-propionamide(0.35 g, 67% yield): mp, 163-165° C.; ¹HNMR (CDCl₃): δ1.44 (t, J=7.0 Hz,3H, OCH₂CH₃), 2.25 (s, 3H, CH₃CO), 2.90 (s, 3H, NCH₃), 2.98 (dd, J=5, 15Hz, 1H, CH₂), 3.05 (s, 4H, NCH₃+CH₂), 3.91 (dd, J=10, 15 Hz, 1H, CH₂),4.09 (q, J=7.0 Hz, 2H, OCH₂CH₃), 5.81-5.87 (m, 1H, CHN), 6.53 (t,J_(H-F)=75 Hz, 1H, CF₂H), 7.11-7.17 (m, 3H, Ar), 7.45 (d, J=7.0 Hz, 1H,Ar), 7.62 (t, J=8.0 Hz, 1H, Ar), 8.73 (d, J=8.8 Hz, 1H, Ar), 9.53 (s,1H, NHCO). ¹³CNMR (DMSO-d₆): δ 14.5, 24.8, 34.8, 35.5, 37.1, 51.3, 64.7,111.9, 113.8, 115.4, 116.1, 117.9, 120.1, 120.2, 122.7, 124.7, 131.3,135.8, 137.5, 138.1, 150.6, 168.1, 169.2, 169.3, 170.0; Anal Calcd forC₂₄H₂₅F₂N₃O₆: C, 58.89; H. 5.15; N, 8.58. Found: C, 58.53; H, 4.78; N,8.51.

5.33 Example 333-(4-Acetylamino-1,3-dioxo-1,3-dihydro-isoindol-2-yl)-3-(4-difluoromethoxy-3-ethoxy-phenyl)-N-hydroxy-propionamide

3-(4-Acetylamino-1,3-dioxo-1,3-dihydro-isoindol-2-yl)-3-(4-difluoromethoxy-3-ethoxy-phenyl)-N-hydroxy-propionamidewas prepared by the procedure of example 8 from3-(4-acetylamino-1,3-dioxo-1,3-dihydro-isoindol-2-yl)-3-(4-difluoromethoxy-3-ethoxy-phenyl)-propionicacid (0.5 g, 1.1 mmol), CDI (0.2 g, 1.3 mmol) and hydroxylaminehydrocloride (0.1 g, 1.4 mmol) in THF (10 ml) to give3-(4-acetylamino-1,3-dioxo-1,3-dihydro-isoindol-2-yl)-3-(4-difluoromethoxy-3-ethoxy-phenyl)-N-hydroxy-propionamideas white solid (0.25 g, 50% yield): mp 148-150° C.; ¹HNMR (CDCl₃): δ1.44(t, J=7.0 Hz, 3H, OCH₂CH₃), 2.19 (s, 3H, CH₃CO), 3.19 (dd, J=5, 15 Hz,1H, CH₂), 3.47 (dd, J=10, 15 Hz, 1H, CH₂), 4.02 (q, J=7.0 Hz, 2H,OCH₂CH₃), 5.75-5.82 (m, 1H, CHN), 6.52 (t, J_(H-F)=75 Hz, 1H, CF₂H),7.02-7.07 (m, 3H, Ar), 7.36 (d, J=7.5 Hz, 1H, Ar), 7.54 (t, J=7.5 Hz,1H, Ar), 8.64 (d, J=8.8 Hz, 1H, Ar), 9.40 (s, 1H, NHCO). ¹³CNMR (CDCl₃):δ 14.5, 24.8, 34.6, 51.1, 64.7, 111.8, 113.5, 115.1, 115.9, 120.1,122.6, 124.9, 131.0, 136.0, 136.7, 137.4, 140.0, 150.5, 167.1, 167.7,169.3, 169.6 Anal Calcd for C₂₂H₂₁F₂N₃O₇+0.3 H₂O: C, 54.73; H, 4.51; N,8.70. Found: C, 54.36; H, 4.25; N, 8.54, 0.1% H₂O.

5.34 Example 341-(4-Difluoromethoxy-3-ethoxy-phenyl)-2-methanesulfonyl-ethylamine

To the THF solution (100 ml) of 4-difluoromethoxy-3-ethoxy-benzaldehyde(21.0 g, 0.1 mol) was added dropwise LiN(TMS)₂ (1M in THF, 100 ml, 0.1mol) at 0° C. After 15 minutes of stirring, BF₃ THF complex (22 ml, 0.2mol) was added to the reaction mixture (1). To the THF solution (100 ml)of methyl sulfone (9.4 g, 0.1 mol) was added LiN(TMS)₂ (1M in THF, 100ml, 0.1 mol) at −78° C. The mixture (2) was stirred at −78° C. for 1hour. Mixture (1) was added to the mixture (2) via a 2-way needle. Themixture was allowed to warm to room temperature and stirred at roomtemperature for 18 hours. MeOH (20 ml) was added to quench the reaction.The mixture was concentrated in vacuo until there was one-fourth left.To the resulted mixture was added HCl aqueous solution (20%, 150 ml)followed by concentrated HCl until pH=3. The mixture was extracted withether (100 ml×3). The aqueous layer was neutralized with NaOH (15 M)till pH=8. The mixture was extracted with methylene chloride (100 ml×3).The combined organic layer was washed with water (100 ml×2), brine (100ml), dried over Na₂SO₄ and concentrated in vacuo. The resulted oil waspurified by silica gel column to give1-(4-difluoromethoxy-3-ethoxy-phenyl)-2-methanesulfonyl-ethylamine (2.8g, 10%) as yellow oil. ¹H NMR (CDCl₃) δ 1.41 (t, J=6.8 Hz, 3H, OCH₂CH₃),3.30-3.40 (m, 2H, CH₂SO₂), 3.45 (s, 3H, CH₃), 4.58-4.64 (m, 1H, CHN),6.53 (t, J=75 Hz, 1H, CF₂H), 6.84-7.12 (m, 3H, Ar).

5.35 Example 35 Cyclopropanecarboxylic acid{2-[1-(4-difluoromethoxy-3-ethoxy-phenyl)-2-methanesulfonyl-ethyl]-3-oxo-2,3-dihydro-1H-isoindol-4-yl}-amide

To the DMF solution (20 ml) of1-(4-difluoromethoxy-3-ethoxy-phenyl)-2-methanesulfonyl-ethylamine (0.80g, 2.7 mmol) was added Et₃N (1.6 ml, 12 mmol) followed by2-bromomethyl-6-(cyclopropanecarbonyl-amino)-benzoic acid methyl ester(1.0 g, 3.2 mmol). The mixture was heated at 90° C. for 12 hours thencooled to room temperature. The mixture was extracted with EtOAc (50 ml)and water (50 ml). The organic layer was washed with water (50 ml) andbrine (50 ml), dried over Na₂SO₄ and concentrated in vacuo. The resultedoil was purified by silica gel chromatography to givecyclopropanecarboxylic acid{2-[1-(4-difluoromethoxy-3-ethoxy-phenyl)-2-methanesulfonyl-ethyl]-3-oxo-2,3-dihydro-1H-isoindol-4-yl}-amideas white solid (0.35 g, 30%): mp 115-117° C.; ¹H NMR (CDCl₃) δ 0.83-0.92(m, 2H, c-CH₂), 1.09-1.13 (m, 2H, c-CH₂), 1.45 (t, J=7.0 Hz, 3H, CH₃),1.66-1.70 (m, 1H, c-CH), 2.98 (s, 3H, SO₂CH₃), 3.64 (dd, J=3.8, 14 Hz,1H, CH₂), 4.10 (q, J=6.8 Hz, 2H, OCH₂), 4.33 (dd, J=10, 14 Hz, 1H, CH₂),4.37 (dd, J=5, 50 Hz, 2H, CH₂N), 5.69-5.74 (m, 1H, NCH), 6.57 (t,J_(H-F)=75 Hz, 1H, CF₂H), 6.94-7.04 (m, 3H, Ar), 7.18 (d, J=7.5 Hz, 1H,Ar), 7.47 (t, J=7.5 Hz, 1H, Ar), 8.45 (d, J=8.5 Hz, 1H, Ar), 10.42 (s,1H, NH); ¹³C NMR (CDCl₃) δ 9.06, 15.3, 16.9, 42.4, 49.0, 52.5, 56.3,65.6, 112.4, 113.9, 116.6, 117.5, 117.6, 118.7, 119.9, 120.7, 123.8,134.5, 136.5, 138.9, 141.1, 141.9, 151.9, 170.9, 173.4; Anal Calcd forC₂₄H₂₆F₂N₂O₆S: C, 56.68; H, 5.15; N. 5.51. Found: C, 56.72; H, 5.15; N.5.38.

5.36 Example 36N-{2-[1-(4-(4-Difluoromethoxy-3-ethoxy-phenyl)-2-methanesulfonyl-ethyl]-1,3-dioxo-2,3-dihydro-1H-isoindol-4-yl}-acetamide

N-{2-[1-(4-Difluoromethoxy-3-ethoxy-phenyl)-2-methanesulfonyl-ethyl]-1,3-dioxo-2,3-dihydro-1H-isoindol-4-yl}-acetamidewas prepared by the procedure of example 4 from1-(4-difluoromethoxy-3-ethoxy-phenyl)-2-methanesulfonyl-ethylamine (0.6g, 0.2 mmol), 3-acetamido-phthalic anhydride (0.4 g, 0.2 mmol) andsodium acetate (0.16 g, 0.2 mmol) in acetic acid (15 ml) to giveN-{2-[1-(4-difluoromethoxy-3-ethoxy-phenyl)-2-methanesulfonyl-ethyl]-1,3-dioxo-2,3-dihydro-1H-isoindol-4-yl}-acetamideas white solid (0.4 g, 40%): mp 118-120° C.; ¹H NMR (CDCl₃) δ1.45 (t,J=7.0 Hz, 3H, CH₃), 2.27 (s, 3H, CH₃CO), 2.92 (s, 3H, SO₂CH₃), 3.66 (dd,J=3.8, 14 Hz, 1H, CH₂), 4.11 (q, J=6.8 Hz, 2H, OCH₂), 5.88-5.94 (m, 1H,NCH), 6.55 (t, J_(H-F)=75 Hz, 1H, CF₂H), 7.12-7.16 (m, 3H, Ar), 7.49 (d,J=7.5 Hz, 1H, Ar), 7.66 (t, J=7.5 Hz, 1H, Ar), 8.77 (d, J=8.5 Hz, 1H,Ar), 9.44 (s, 1H, NH); ¹³C NMR (CDCl₃) δ 14.5, 24.9, 41.7, 48.3, 54.1,64.9, 11.5, 113.6, 115.0, 115.8, 118.3, 120.0, 120.2, 123.1, 125.1,130.9, 135.4, 136.2, 137.7, 140.4, 150.9, 167.4, 169.1, 169.4; AnalCalcd for C₂₂H₂₂F₂N₂O₇S: C, 53.22; H, 4.47; N, 5.64. Found: C, 53.18; H,4.20; N, 5.64.

5.37 Example 37 Cyclopropanecarboxylic acid{2-[2-carbamoyl-1-(4-difluoromethoxy-3-ethoxy-phenyl)-ethyl]-7-chloro-3-oxo-2,3-dihydro-1H-isoindol-4-yl}-amide

Cyclopropanecarboxylic acid{2-[2-carbamoyl-1-(4-difluoromethoxy-3-ethoxy-phenyl)-ethyl]-7-chloro-3-oxo-2,3-dihydro-1H-isoindol-4-yl}-amidewas prepared by the procedure of Example 6 from3-[4-chloro-7-(cyclopropanecarbonyl-amino)-1-oxo-1,3-dihydro-isoindol-2-yl]-3-(4-difluoromethoxy-3-ethoxy-phenyl)-propionicacid (0.55 g, 1.1 mmol), CDI (0.26 g, 1.6 mmol) and NH₄OH (0.35 ml, 3.3mmol) in THF (15 ml) to give cyclopropanecarboxylic acid{2-[2-carbamoyl-1-(4-difluoromethoxy-3-ethoxy-phenyl)-ethyl]-7-chloro-3-oxo-2,3-dihydro-1H-isoindol-4-yl}-amideas white solid (0.10 g, 20%): mp 198-200° C.; ¹HNMR (CDCl₃): δ 0.89-0.93(m, 2H, c-CH₂), 1.10-1.20 (m, 2H, c-CH₂), 1.44 (t, J=7.5 Hz, 3H,OCH₂CH₃), 1.66-1.68 (m, 1H, c-CH), 2.98 (dd, J=5, 15 Hz, 1H, CH₂), 3.43(dd, J=10, 15 Hz, 1H, CH₂), 4.09 (q, J=7.5 Hz, 2H, OCH₂CH₃), 4.18 (d,J=17.5 Hz, 1H, CHHN), 4.37 (d, J=17.5 Hz, 1H, CHHN), 5.48 (br, 1H, NHH),5.57-5.53 (m, 1H, CHN), 5.92 (br, 1H, NHH), 6.86 (t, J_(H-F)=75 Hz, 1H,CF₂H), 6.94-7.02 (m, 3H. Ar), 7.16 (d, J=10 Hz, 1H, Ar), 7.38 (t, J=8Hz, 1H, Ar), 8.45 (d, J=8 Hz, 1H, Ar), 10.40 (s, 1H, NHCO). ¹³CNMR(CDCl₃): δ 8.5, 14.6, 16.2, 38.6, 47.9, 54.1, 64.9, 101.57, 102.3,113.2, 116.0, 119.2, 119.8, 121.8, 123.0, 133.2, 136.8, 137.1, 139.1,150.9, 169.4, 171.1, 172.6; Anal Calcd for C₂₄H₂₆ClF₂N₃O₅; C, 56.75; H.4.76; N. 8.27. Found: C, 56.68; H, 4.63; N, 8.04.

5.38 Example 38N-{2-[1-(4-Difluoromethoxy-3-ethoxy-phenyl)-3-morpholin-4-yl-3-oxo-propyl]-1,3-dioxo-2,3-dihydro-1H-isoindol-4-yl}-acetamide

To a solution of3-(4-acetylamino-1,3-dioxo-1,3-dihydro-isoindol-2-yl)-3-(4-difluoromethoxy-3-ethoxy-phenyl)-propionicacid (400 mg, 0.86 mmol) in tetrahydrofurane was addedcarbonyldiimidazole (160 mg, 1 mmol) at room temperature. The solutionwas stirred for 2 hours at room temperature. To the mixture was addedmorpholine (0.12 ml, 1.3 mmol). The resulted mixture was stirred at roomtemperature for 2 hours. Water (5 ml) was added to the reaction mixture.THF was removed in vacuo and the resulted mixture was taken up in ethylacetate (20 ml). The organic layer was washed with saturated sodiumbicarbonate solution (3×10 ml), water (10 ml), brine (10 ml) and wasdried over magnesium sulfate. The solvent was removed in vacuo. Theresulted oil was purified by HPLC (acetonitrile: water=45:55) to giveN-{2-[1-(4-difluoromethoxy-3-ethoxy-phenyl)-3-morpholin-4-yl-3-oxo-propyl]-1,3-dioxo-2,3-dihydro-1H-isoindol-4-yl}-acetamideas a white solid (200 mg, 44%): mp, 109-111° C.; ¹HNMR (CDCl₃): 1.44 (t,J=7.5 Hz, 3H, OCH₂CH₃), 2.26 (s, 3H, CH₃CO), 3.02 (dd, J=5, 17.5 Hz, 1H,CHHCO), 3.49-3.68 (m, 8H, morpholine ring), 3.9 (dd, J=7.5, 17.5 Hz, 1H,CHHCO), 4.09 (q, J=7.5 Hz, 2H, OCH₂CH₃), 5.84 (dd, J=5, 10 Hz, 1H, CHN),6.54 (t, J_(H-F)=75 Hz, 1H, OCF₂H), 7.11-7.16 (m, 3H, Ar), 7.45 (d,J=7.5 Hz, 1H, Ar), 7.63 (t, J=7.5 Hz, 1H, Ar), 8.75 (d, J=7.5 Hz, 1H,Ar), 9.50 (s, 1H, NHCO). ¹³C NMR (CDCl₃): 15.3, 25.6, 35.1, 42.7, 46.6,51.9, 65.5, 67.2, 67.5, 112.6, 114.4, 115.9, 116.7, 118.6, 120.8, 120.9,123.5, 125.5, 131.9, 136.6, 138.2, 138.5, 140.6, 151.3, 168.6, 168.7,169.9, 170.6; Anal. Calcd. for C₂₆H₂₇F₂N₃O₇: C, 58.75; H, 5.12; N, 7.91.Found: C, 58.48; H, 5.09; N, 7.79;

5.39 Example 39N-{2-[1-(4-Difluoromethoxy-3-ethoxy-phenyl)-3-morpholin-4-yl-3-oxo-propyl]-3-oxo-2,3-dihydro-1H-isoindol-4-yl}-acetamide

To a solution of3-(7-acetylamino-1-oxo-1,3-dihydro-isoindol-2-yl)-3-(4-difluoromethoxy-3-ethoxy-phenyl)-propionicacid (300 mg, 0.67 mmol) in tetrahydrofurane was addedcarbonyldiimidazole (130 mg, 0.80 mmol) at room temperature. Thesolution was stirred for 2 hours at room temperature. To the mixture wasadded morpholine (0.1 ml, 1.0 mmol). The resulted mixture was stirred atroom temperature for 2 hours. Water (5 ml) was added to the reactionmixture. THF was removed in vacuo and the resulted mixture was taken upin ethyl acetate (20 ml). The organic layer was washed with saturatedsodium bicarbonate solution (3×10 ml), water (10 ml), brine (10 ml) andwas dried over magnesium sulfate. The solvent was removed in vacuo. Theresulted oil was purified by HPLC (acetonitrile: water=45:55) to giveN-{2-[1-(4-difluoromethoxy-3-ethoxy-phenyl)-3-morpholin-4-yl-3-oxo-propyl]-3-oxo-2,3-dihydro-1H-isoindol-4-yl}-acetamideas a white solid (250 mg, 72%): mp, 164-166° C.; ¹HNMR (CDCl₃): 1.43 (t,J=7.5 Hz, 3H, OCH₂CH₃), 2.25 (s, 3H, CH₃CO), 3.04 (dd, J=6, 10 Hz, 1H,CHHCO), 3.54-3.72 (m, 9H, morpholine ring+CHHCO), 4.08 (q, J=7.5 Hz, 2H,OCH₂CH₃), 4.27 (d, J=17 Hz, 1H, CHHN), 4.44 (d, J=17 Hz, 1H, CHHN),5.54-5.59 (m, 1H, CHN), 6.85 (t, H_(H-F)=75 Hz, 1H, OCF₂H), 6.94-7.16(m, 3H, Ar), 7.14 (d, J=8 Hz, 1H, Ar), 7.46 (t, J=7.5 Hz, 1H, Ar), 8.45(d, J=7.5 Hz, 1H, Ar), 10.60 (s, 1H, NHCO). ¹³C NMR (CDCl₃): 15.3, 25.6,36.5, 42.8, 46.9, 49.8, 54.7, 65.5, 67.2, 67.4, 112.6, 114.1, 116.7,117.6, 118.3, 118.4, 119.9, 120.8, 123.5, 133.9, 138.5, 140.5, 142.2,151.5, 166.7, 169.7, 170.4; Anal. Calcd. for C₂₆H₂₉F₂N₃O₆: C, 60.34; H,5.65; N, 8.12. Found: C, 60.04; H, 5.71; N, 8.19.

5.40 Example 403-(4-Acetylamino-1,3-dioxo-1,3-dihydro-isoindol-2-yl)-3-(3,4-bis-difluoromethoxy-phenyl)-N,N-dimethyl-propionamide

To a solution of3-(4-acetylamino-1,3-dioxo-1,3-dihydro-isoindol-2-yl)-3-(3,4-bis-difluoromethoxy-phenyl)-propionicacid (350 mg, 0.72 mmol) in tetrahydrofurane was addedcarbonyldiimidazole (175 mg, 1.08 mmol) at room temperature. Thesolution was stirred for 2 hours at room temperature. To the mixture wasadded dimethylamine in THF (0.73 ml, 1.45 mmol). The resulted mixturewas stirred at room temperature for 2 hours. Water (5 ml) was added tothe reaction mixture. THF was removed in vacuo and the resulted mixturewas taken up in ethyl acetate (20 ml). The organic layer was washed withsaturated sodium bicarbonate solution (3×10 ml), water (10 ml), brine(10 ml) and dried over magnesium sulfate. The solvent was removed invacuo. The resulted oil was purified by HPLC (acetonitrile: water=45:55)to give3-(4-acetylamino-1,3-dioxo-1,3-dihydro-isoindol-2-yl)-3-(3,4-bis-difluoromethoxy-phenyl)-N,N-dimethyl-propionamideas a white solid (200 mg, 55%): mp, 83-85° C.; ¹HNMR (CDCl₃): 2.25 (s,3H, CH₃CO), 2.91 (s, 3H, NCH₃), 3.06-3.13 (m, 4H, CHHCO+NCH₃), 3.85 (dd,J=10, 15 Hz, 1H, CHHCO), 5.85-5.91 (m, 1H, CHN), 6.80 (t, J_(H-F)=75 Hz,1H, OCF₂H), 6.84 (t, J_(H-F)=75 Hz, 1H, OCF₂H), 7.23 (d, J=7.5 Hz, 1H,Ar), 7.41-7.47 (m, 3H, Ar), 7.63 (t, J=7.5 Hz, 1H, Ar), 8.72 (d, J=7.5Hz, 1H, Ar), 9.51 (s, 1H, NHCO). ¹³C NMR (CDCl₃): 24.8, 34.5, 35.4,36.9, 50.5, 111.4, 111.5, 115.2, 115.6, 115.7, 117.9, 119.8, 119.9,122.0, 122.4, 124.7, 126.1, 131.1, 135.8, 137.4, 138.2, 141.9, 142.2,167.7, 168.8, 169.1, 169.7; Anal. Calcd. for C₂₃H₂₁F₄N₃O₆: C, 54.02; H,4.14; N, 8.22. Found: C, 53.89; H, 3.88; N, 8.13.

5.41 Example 413-(3,4-Bis-difluoromethoxy-phenyl)-3-[4-chloro-7-(cyclopropanecarbonyl-amino)-1-oxo-1,3-dihydro-isoindol-2-yl]-propionicacid methyl ester

To a solution of 3-amino-3-(3,4-bis-difluoromethoxy-phenyl)-propionicacid methyl ester (50 mg, 0.16 mmol) and triethyl amine (0.09 ml, 3.1mmol) in DMF (2 ml) was added2-bromomethyl-3-chloro-6-(cyclopropanecarbonyl-amino)-benzoic acidmethyl ester (67 mg, 0.19 mmol). The mixture was heated at 90° C. undernitrogen atmosphere overnight. The solvent was removed in vacuo. Theresulted oil was extracted with ethyl acetate (50 ml) and 1N HCl (50ml). The organic layer was washed with water (30 ml), brine (30 ml) anddried over magnesium sulfate. The solvent was removed in vacuo and theresulted oil was purified by silica gel chromatography (hexane:ethylacetate=2:1) to give3-(3,4-bis-difluoromethoxy-phenyl)-3-[4-chloro-7-(cyclopropanecarbonyl-amino)-1-oxo-1,3-dihydro-isoindol-2-yl]-propionicacid methyl ester as a off-white solid (15 mg, 17%). mp, 175-177° C.;¹HNMR (CDCl₃): δ 0.88-0.94 (m, 2H, cyclopropyl ring CH₂), 1.07-1.13 (m,2H, cyclopropyl ring CH₂), 1.65-1.68 (m, 1H, cyclopropyl ring CH), 3.14(dd, J=6, 15 Hz, 1H, CHHCO), 3.28 (dd, J=10, 15 Hz, 1H, CHHCO), 3.70 (s,3H, OCH₃), 4.13 (d, J=17 Hz, 1H, CHHN), 4.36 (d, J=17 Hz, 1H, CHHN),5.79-5.85 (m, 1H, CHN). 6.23 (t, J_(H-F)=75 Hz, 1H, OCF₂H), 6.26 (t,J_(H-F)=75 Hz, 1H, OCF₂H), 7.28-7.41 (m, 4H, Ar), 8.45 (d, J=9 Hz, 1H.Ar), 10.38 (s, 1H, NHCO); ¹³C NMR (CDCl₃): 8.45, 15.25, 16.16, 36.46,46.34, 51.62, 52.31, 65.38, 111.38, 111.42, 115.57, 115.61, 118.64,119.76, 119.80, 121.76, 121.81, 122.77, 125.45, 133.14, 136.89, 137.03,138.86, 142.29, 142.21, 168.79, 170.20, 172.73; Anal. Calcd. forC₂₄H₂₁ClF₄N₂O₆: C, 52.90; H, 3.88; N, 5.14. Found: C, 52.78; H, 3.80; N,5.01;

5.42 Example 42 Cyclopropanecarboxylic acid {2-[1-(3,4-bis-difluoromethoxy-phenyl)-2-dimethylcarbamoyl-ethyl]-3-oxo-2,3-dihydro-1H-isoindol-4-yl}-amide

To a solution of3-(3,4-bis-difluoromethoxy-phenyl)-3-[7-(cyclopropanecarbonyl-amino)-1-oxo-1,3-dihydro-isoindol-2-yl]-propionicacid (400 mg, 0.81 mmol) in tetrahydrofurane (10 ml) was addedcarbonyldiimidazole (200 mg, 1.21 mmol) at room temperature. Thesolution was stirred at room temperature for 2 hours. To the mixture wasadded dimethylamine in THF (0.8 ml, 1.6 mmol). The resulted mixture wasstirred at room temperature for 3 hours. Water (20 ml) was added to thereaction mixture. The solvent was removed in vacuo. The resulted mixturewas taken up in ethyl acetate (30 ml) and washed with water (20 ml). Theorganic layer was washed with saturated sodium bicarbonate solution(3×20 ml), water (20 ml), brine (20 ml) and dried over magnesiumsulfate. The solvent was removed in vacuo. The resulted oil was stirredwith ether overnight. The suspension was filtered to give a yellowishsolid. The solid was purified by HPLC (CH₃CN: Water=45:55) to givecyclopropanecarboxylic acid{2-[1-(3,4-bis-difluoromethoxy-phenyl)-2-dimethylcarbamoyl-ethyl]-3-oxo-2,3-dihydro-1H-isoindol-4-yl}-amideas a white solid (200 mg, 50%): mp, 158-160° C.; ¹H NMR (CDCl₃): δ0.86-0.92 (m, 2H, cyclopropyl ring CH₂), 1.08-1.12 (m, 2H, cyclopropylring CH₂), 1.66-1.70 (m, 1H, cyclopropyl ring CH), 2.93 (s, 3H, NCH₃),3.04 (dd, J=5, 15 Hz, 1H, CHHCO), 3.10 (s, 3H, NCH₃), 3.64 (dd, J=8, 15Hz, 1H, CHHCO), 4.13 (t, J=15 Hz, 2H, CHHN), 5.56-5.62 (m, 1H, CHN),6.51 (t, J_(H-F)=75 Hz, 1H, OCF₂H), 6.54 (t, J_(H-F)=75 Hz, 1H, OCF₂H),7.01 (d. J=8 Hz, 1H, Ar), 7.23-7.36 (m, 31-1, Ar), 7.44 (t, J=8 Hz, 1H,Ar), 8.43 (d, J=8 Hz, 1H, Ar), 10.52 (s, 1H, NHCO); ¹³C NMR (CDCl₃):8.22, 16.16, 35.58, 35.92, 37.27, 49.59, 53.83, 58.94, 111.0, 115.65,117.64, 117.67, 119.90, 119.80, 121.66, 122.58, 125.64, 133.27, 138.05,138.69, 141.49, 169.11, 169.81, 172.65; Anal. Calcd. for C₂₅H₂₅F₄N₃O₅:C, 57.36; H, 4.81; N, 8.03. Found: C, 57.22; H, 4.76; N, 8.11.

5.43 Example 43 Cyclopropanecarboxylic acid {2-[1-(3,4-bis-difluoromethoxy-phenyl)-2-carbamoyl-ethyl]-3-oxo-2,3-dihydro-1H-isoindol-4-yl}-amide

To a solution of3-(3,4-bis-difluoromethoxy-phenyl)-3-[7-(cyclopropanecarbonyl-amino)-1-oxo-1,3-dihydro-isoindol-2-yl]-propionicacid (400 mg, 0.81 mmol) in tetrahydrofurane was addedcarbonyldiimidazole (200 mg, 1.21 mmol) at room temperature. Thesolution was stirred for 2 hours at room temperature. To the mixture wasadded ammonium hydroxide (0.12 ml, 1.6 mmol). The resulted mixture wasstirred at room temperature for 2 hours. Water (5 ml) was added to thereaction mixture. THF was removed in vacuo and the resulted mixture wastaken up in ethyl acetate (20 ml). The organic layer was washed withsaturated sodium bicarbonate solution (3×10 ml), water (10 ml), brine(10 ml) and was dried over magnesium sulfate. The solvent was removed invacuo. The resulted oil was purified by HPLC (acetonitrile: water=45:55)to give cyclopropanecarboxylic acid{2-[1-(3,4-bis-difluoromethoxy-phenyl)-2-carbamoyl-ethyl]-3-oxo-2,3-dihydro-1H-isoindol-4-yl}-amideas a white solid (230 mg, 46%): mp, 198-200° C.; ¹HNMR (DMSO-d₆): δ 0.87(d, J=5 Hz, 4H, cyclopropyl ring CH₂CH₂), 1.74-1.79 (m, 1H, cyclopropylring CH), 2.99 (d, J=7.5 Hz, 2H, CH₂CONH₂), 4.29 (d, J=18 Hz, 1H, CHHN),4.61 (d, J=18 Hz, 1H, CHHN), 5.69-5.75 (m, 1H, CHN), 6.97 (broad, 1H,NM), 7.19 (t, J_(H-F)=75 Hz, 1H, OCF₂H), 7.22 (t, J_(H-F)=75 Hz, 1H,OCF₂H), 7.34-7.37 (m, 3H, Ar), 7.47-7.58 (m, 2H, NHH+ 1Ar), 8.23 (d, J=8Hz, 1H, Ar), 10.51 (s, 1H, NHCO); ¹³C NMR (CDCl₃): 8.26, 15.95, 38.10,47.37, 51.89, 112.77, 112.90, 116.90, 117.03, 117.40, 117.70, 117.99,120.57, 121.03, 121.16, 121.74, 125.72, 133.38, 137.51, 138.81, 141.60,141.65, 141.70, 142.15, 142.20, 142.25, 142.79, 168.74, 171.37, 172.24;Anal. Calcd. for C₂₃H₂₁F₄N₃O₅: C, 55.76; H, 4.27; N, 8.48. Found: C,55.98; H, 4.00; N, 8.46.

5.44 Example 44 Cyclopropanecarboxylic acid {2-[1-(3,4-bis-difluoromethoxy-phenyl)-2-hydroxycarbamoyl-ethyl]-3-oxo-2,3-dihydro-1H-isoindol-4-yl}-amide

To a solution of3-(3,4-bis-difluoromethoxy-phenyl)-3-[7-(cyclopropanecarbonyl-amino)-1-oxo-1,3-dihydro-isoindol-2-yl]-propionicacid (400 mg, 0.81 mmol) in tetrahydrofurane was addedcarbonyldiimidazole (200 mg, 1.21 mmol) at room temperature. Thesolution was stirred for 2 hours at room temperature. To the mixture wasadded hydroxylamine (120 mg, 1.6 mmol). The resulted mixture was stirredat room temperature for 2 hours. Water (5 ml) was added to the reactionmixture. THF was removed in vacuo and the resulted mixture was taken upin ethyl acetate (20 ml). The organic layer was washed with saturatedsodium bicarbonate solution (3×10 ml), water (10 ml), brine (10 ml) anddried over magnesium sulfate. The solvent was removed in vacuo. Theresulted oil was purified by HPLC (acetonitrile: water=45:55) to givecyclopropanecarboxylic acid{2-[1-(3,4-bis-difluoromethoxy-phenyl)-2-hydroxycarbamoyl-ethyl]-3-oxo-2,3-dihydro-1H-isoindol-4-yl}-amideas a white solid (230 mg, 46%): mp, 191-193° C.; ¹HNMR (CDCl₃): δ0.88-0.93 (m, 2H, cyclopropyl ring CH₂), 1.09-1.12 (m, 2H, cyclopropylring CH₂), 1.64-1.67 (m, 1H, cyclopropyl ring CH), 2.84-2.92 (m, 1H,CHHCO), 3.38-3.48 (m, 1H, CHHCO), 4.18 (d, J=18 Hz, 1H, CHHN), 4.36 (d,J=18 Hz, 1H, CHHN), 5.47-5.49 (m, 1H, CHN), 6.52 (t, J_(H-F)=75 Hz, 1H,OCF₂H), 6.54 (t, J_(H-F)=75 Hz, 1H, OCF₂H), 6.99 (d, J=8 Hz, 1H, Ar),7.25-7.31 (m. 3H, Ar), 7.45 (t, J=8 Hz, 1H, Ar), 8.43 (d, J=8 Hz, 1H,Ar), 9.20 (broad, 1H, NHOH), 10.35 (s, 1H, NHCO); ¹³C NMR (DMSO): 30.82,35.26, 38.47, 46.17, 47.50, 51.68, 112.50, 116.83, 117.01, 117.39,117.66, 117.99, 120.56, 120.97, 121.73, 125.73, 133.41, 137.47, 138.38,142.76, 146.83, 166.12, 168.68, 172.21, 179.98. Anal. Calcd. forC₂₃H₂₁F₄N₃O₆: C, 54.02; H, 4.14; N, 8.22. Found: C, 53.89; H, 3.96; N,8.22.

5.45 Example 453-(3,4-Bis-difluoromethoxy-phenyl)-3-[7-(cyclopropanecarbonyl-amino)-1-oxo-1,3-dihydro-isoindol-2-yl]-propionicacid

To a solution of3-(3,4-bis-difluoromethoxy-phenyl)-3-[7-(cyclopropanecarbonyl-amino)-1-oxo-1,3-dihydro-isoindol-2-yl]-propionicacid methyl ester (2.0 g, 4 mmol) in tetrahydrofurane (20 ml) was addedNaOH (0.8 ml of 10 N) at room temperature. The solution was stirredovernight at room temperature. The resulted suspension was filtered toget a white solid. The solid was dissolved in water (30 ml) and addedconc. HCl dropwise until the pH is about 4. The resulted mixture wasextracted with CH₂Cl₂ (3×40 ml). The organic layer was dried overNa₂SO₄, filtered and concentrated to give3-(3,4-bis-difluoromethoxy-phenyl)-3-[7-(cyclopropanecarbonyl-amino)-1-oxo-1,3-dihydro-isoindol-2-yl]-propionicacid as a white solid (1.4 g, 70%): mp, 94-96° C.; ¹HNMR (CDCl₃) δ0.86-0.93 (m, 2H, cyclopropyl ring CH₂), 1.06-1.12 (m, 2H, cyclopropylring CH₂), 1.65-1.71 (m, 1H, cyclopropyl ring CH), 3.13 (dd, J=6, 16 Hz,1H, CHHCO), 3.32 (dd, J=9, 16 Hz, 1H, CHHCO), 4.15 (d, J=15 Hz, 1H,CHHN), 4.36 (d, J=15 Hz, 1H, CHHN), 5.78-5.84 (m, 1H, CHN), 6.21 (t,J_(H-F)=75 Hz, 1H, OCF₂H), 6.24 (t, J_(H-F)=75 Hz, 1H, OCF₂H), 7.01 (d,J=7.5 Hz, 1H, Ar), 7.26-7.28 (m, 3H, Ar), 7.44 (t, J=7.5 Hz, 1H, Ar),8.43 (d, J=7.5 Hz, 1H, Ar), 10.42 (s, 1H, NHCO); ¹³C NMR (CDCl₃): 8.35,8.38, 16.14, 36.55, 47.00, 51.29, 99.24, 111.36, 111.42, 115.60, 116.84,117.02, 118.03, 119.74, 119.79, 121.59, 122.76, 125.46, 133.61, 137.09,138.03, 141.12, 142.21, 142.38, 142.43, 169.63, 173.03, 173.82. Anal.Calcd. for C₂₃H₂₀F₄N₂O₆: C, 55.65; H, 4.06; N, 5.64. Found: C, 55.33; H,3.96; N, 5.38.

5.46 Example 46 50 mg Solid Tablets

Tablets, each containing 50 mg of Cyclopropanecarboxylic acid{2-[2-carbamoyl-1-(4-difluoromethoxy-3-ethoxy-phenyl)-ethyl]-3-oxo-2,3-dihydro-1H-isoindol-4-yl}-amide,can be prepared in the following manner:

Constituents (for 1000 tablets) Cyclopropanecarboxylic acid{2-[2-carbamoyl-1-(4- 50.0 g difluoromethoxy-3-ethoxy-phenyl)-ethyl]-3-oxo-2,3-dihydro-1H-isoindol-4-yl}-amide lactose 50.7 g  wheat starch 7.5 g polyethyleneglycol 6000 5.0 g talc 5.0 g magnesium stearate 1.8 g demineralizedwater q.s.

The solid ingredients are first forced through a sieve of 0.6 mm meshwidth. The active ingredient, lactose, talc, magnesium stearate and halfof the starch then are mixed. The other half of the starch is suspendedin 40 mL of water and this suspension is added to a boiling solution ofthe polyethylene glycol in 100 mL of water. The resulting paste is addedto the pulverulent substances and the mixture is granulated, ifnecessary with the addition of water. The granulate is dried overnightat 35EC, forced through a sieve of 1.2 mm mesh width and compressed toform tablets of approximately 6 mm diameter which are concave on bothsides.

5.47 Example 47 100 mg Solid Tablets

Tablets, each containing 100 mg of Cyclopropanecarboxylic acid{2-[1-(4-difluoromethoxy-3-ethoxy-phenyl)-2-dimethylcarbamoyl-ethyl]-3-oxo-2,3-dihydro-1H-isoindol-4-yl}-amide,can be prepared in the following manner:

Constituents (for 1000 tablets) Cyclopropanecarboxylic acid{2-[1-(4-difluoromethoxy-3- 50.0 gethoxy-phenyl)-2dimethylcarbamoyl-ethyl]-3-oxo-2,3-dihydro-1H-isoindol-4-yl}-amide lactose 100.7 g  wheat starch 47.0 gmagnesium stearate  3.0 g

All the solid ingredients are first forced through a sieve of 0.6 mmmesh width. The active ingredient, lactose, magnesium stearate and halfof the starch then are mixed. The other half of the starch is suspendedin 40 mL of water and this suspension is added to 100 mL of boilingwater. The resulting paste is added to the pulverulent substances andthe mixture is granulated, if necessary with the addition of water. Thegranulate is dried overnight at 35EC, forced through a sieve of 1.2 mmmesh width and compressed to form tablets of approximately 6 mm diameterwhich are concave on both sides.

5.48 Example 48 75 mg Chewable Tablets

Tablets for chewing, each containing 75 mg of Cyclopropanecarboxylicacid{2-[1-(4-difluoromethoxy-3-ethoxy-phenyl)-2-hydroxycarbamoyl-ethyl]-3-oxo-2,3-dihydro-1H-isoindol-4-yl}-amide,can be prepared in the following manner:

Composition (for 1000 tablets) Cyclopropanecarboxylic acid{2-[1-(4-difluoromethoxy-3- 75.0 gethoxy-phenyl)-2-hydroxycarbamoyl-ethyl]-3-oxo-3,3-dihydro-1H-isoindol-4-yl}-amide mannitol 230.0 g  lactose 150.5 g  talc21.0 g glycine 12.5 g stearic acid 10.0 g saccharin 1.5. 5% gelatinsolution q.s.

All the solid ingredients are first forced through a sieve of 0.25 mmmesh width. The mannitol are the lactose are mixed, granulated with theaddition of gelatin solution, forced through a sieve of 2 mm mesh width,dried at 50EC and again forced through a sieve of 1.7 mm mesh width{2-[1-(4-difluoromethoxy-3-ethoxy-phenyl)-2-hydroxycarbamoyl-ethyl]-3-oxo-2,3-dihydro-1H-isoindol-4-yl}-amide,the glycine and the saccharin are carefully mixed, the mannitol, thelactose granulate, the stearic acid and the talc are added and the wholeis mixed thoroughly and compressed to form tablets of approximately 10mm diameter which are concave on both sides and have a breaking grooveon the upper side.

5.49 Example 49 10 mg Tablets

Tablets, each containing 10 mg3-(7-Acetylamino-1-oxo-1,3-dihydro-isoindol-2-yl)-3-(4-difluoromethoxy-3-ethoxy-phenyl)-propionamide,can be prepared in the following manner.

Composition (for 1000 tablets)3-(7-Acetylamino-1-oxo-1,3-dihydro-isoindol-2-yl)-3- 10.0 g(4-difluoromethoxy-3-ethoxy-phenyl)-propionamide lactose 328.5 g  cornstarch 17.5 g polyethylene glycol 6000  5.0 g talc 25.0 g magnesiumstearate  4.0 g demineralized water q.s.

The solid ingredients are first forced through a sieve of 0.6 mm meshwidth. Then the active amide ingredient, lactose, talc, magnesiumstearate and half of the starch are intimately mixed. The other half ofthe starch is suspended in 65 mL and this suspension is added to aboiling solution of the polyethylene glycol in 260 mL of water. Theresulting paste is added to the pulverulent substances, and the whole ismixed and granulated, if necessary with the addition of water. Thegranulate is dried overnight at 35EC, forced through a sieve of 1.2 mmmesh width and compressed to form tablets of approximately 10 mmdiameter which are concave on both sides and have a breaking notch onthe upper side.

5.50 Example 50 100 mg Gelatin Capsules

Gelatin dry-filled capsules, each containing 100 mgCyclopropanecarboxylic acid{2-[1-(4-difluoromethoxy-3-ethoxy-phenyl)-2-dimethylcarbamoyl-ethyl]-3-oxo-2,3-dihydro-1H-isoindol-4-yl}-amide,can be prepared in the following manner:

Composition (for 1000 tablets) Cyclopropanecarboxylic acid{2-[1-(4-difluoromethoxy-3- 100.0 g ethoxy-phenyl)-2-dimethylcarbamoyl-ethyl]-3-oxo-2,3-dihydro-1H-isoindol-4-yl}-amide microcrystalline cellulose 30.0 g sodium lauryl sulfate 2.5 g magnesium stearate 8.0 g

The sodium lauryl sulfate is sieved into the Cyclopropanecarboxylic acid{2-[1-(4-difluoromethoxy-3-ethoxy-phenyl)-2-dimethylcarbamoyl-ethyl]-3-oxo-2,3-dihydro-1H-isoindol-4-yl}-amidethrough a sieve of 0.2 mm mesh width and the two components areintimately mixed for 10 minutes. The microcrystalline cellulose is thenadded through a sieve of 0.9 mm mesh width and the whole is againintimately mixed for 10 minutes. Finally, the magnesium stearate isadded through a sieve of 0.8 mm width and, after mixing for a further 3minutes, the mixture is introduced in portions of 140 mg each into size0 (elongated) gelatin dry-fill capsules.

5.51 Example 51 Injectable Solution

A 0.2% injection or infusion solution can be prepared, for example, inthe following manner:

Cyclopropanecarboxylic acid {2-[1-(4-difluoromethoxy- 5.0 g3-ethoxy-phenyl)-2-dimethylcarbamoyl-ethyl]-3-oxo-2,3-dihydro-1H-isoindol-4-yl}-amide sodium chloride 22.5 g phosphate bufferpH 7.4 300.0 g demineralized water to 2500.0 mL

Cyclopropanecarboxylic acid{2-[1-(4-difluoromethoxy-3-ethoxy-phenyl)-2-dimethylcarbamoyl-ethyl]-3-oxo-2,3-dihydro-1H-isoindol-4-yl}-amideis dissolved in 1000 mL of water and filtered through a microfilter. Thebuffer solution is added and the whole is made up to 2500 mL with water.To prepare dosage unit forms, portions of 1.0 or 2.5 mL each areintroduced into glass ampoules (each containing respectively 2.0 or 5.0mg of amide).

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference. Although the foregoing invention has beendescribed in some detail by way of illustration and example for purposesof clarity of understanding, it will be readily apparent to those ofordinary skill in the art in light of the teachings of this inventionthat certain changes and modifications may be made thereto withoutdeparting from the spirit or scope of the appended claims.

1. A method of inhibiting PDE4 in a mammal comprising administering tosaid mammal an effective amount of a compound having the formula (I):

wherein: Y is —C(O)—, —CH₂, —CH₂C(O)—, —C(O)CH₂—, or SO₂; Z is —H,—C(O)R₃, —(C₀₋₁-alkyl)-SO₂—(C₁₋₄-alkyl), —C₁₋₈-alkyl, —CH₂OH,CH₂(O)(C₁₋₈-alkyl) or —CN; R₁ and R₂ are each independently —CHF₂,—C₁₋₈-alkyl, —C₃₋₁₈-cycloalkyl, or —(C₁₋₁₀-alkyl)(C₃₋₁₈-cycloalkyl), andat least one of R₁ and R₂ is CHF₂; R₃ is —NR₄R₅, -alkyl, —OH, —O-alkyl,phenyl, benzyl, substituted phenyl, or substituted benzyl; R₄ and R₅ areeach independently —H, —C₁₋₈-alkyl, —OH, —OC(O)R₆; R₆ is —C₁₋₈-alkyl,-amino(C₁₋₈-alkyl), -phenyl, -benzyl, or -aryl; X₁, X₂, X₃, and X₄ areeach independent —H, -halogen, -nitro, —NH₂, —CF₃, —C₁₋₆-alkyl,—(C₀₋₄-alkyl)-(C₃₋₆-cycloalkyl), (C₀₋₄-alkyl)-NR₇R₈,(C₀₋₄-alkyl)-N(H)C(O)-(R₈), (C₀₋₄-alkyl)-N(H)C(O)N(R₇R₈),(C₀₋₄-alkyl)-N(H)C(O)O(R₇R₈), (C₀₋₄-alkyl)-OR₈, (C₀₋₄-alkyl)-imidazolyl,(C₀₋₄-alkyl)-pyrrolyl, (C₀₋₄-alkyl)-oxadiazolyl, or(C₀₋₄-alkyl)-triazolyl or X₁ and X₂ or X₂ and X₃ or X₃ and X₄ are takentogether with the atoms that join them to form a cycloalkyl orheterocycloalkyl ring of 3, 4, 5, 6 or 7 atoms; and R₇ and R₈ are eachindependently H, C₁₋₉-alkyl, C₃₋₆-cycloalkyl,(C₁₋₆-alkyl)-(C₃₋₆-cycloalkyl), (C₁₋₆-alkyl)-N(R₇R₈), (C₁₋₆-alkyl)-OR₈,phenyl, benzyl, or aryl; or a pharmaceutically acceptable salt,stereoisomer, or prodrug thereof.
 2. The method of claim 1, wherein theeffective amount of the compound of formula (I) is from about 0.1 mg toabout 300 mg per day.
 3. The method of claim 2, wherein the effectiveamount of the compound of formula (I) is from about 1 mg to about 250 mgper day.
 4. The method of claim 1, wherein the compound of formula (I)is administered orally, parenterally, topically or mucosally.
 5. Themethod of claim 1, wherein the mammal or mammalian cell is human.